SK7222000A3 - Combination of an aldose reductase inhibitor and a glycogen phosphorylase inhibitor - Google Patents

Abstract

Pharmaceutical combination compositions and methods including aldose reductase inhibitors and glycogen phosphorylase inhibitors. The compositions and methods are useful for the treatment insulin resistant conditions such as diabetes and in reducing tissue damage due to ischemia.

Description

Technical field

The invention relates to a pharmaceutical combination of an aldose reductase inhibitor and a glycogen phosphorylase inhibitor and kits comprising such a combination. Furthermore, the invention relates to the use of this combination for the treatment of diabetes, hyperglycemia, hypercholesterolemia, hypertension, hyperinsulinemia, hyperlipidemia, atherosclerosis and tissue ischemia in mammals.

BACKGROUND OF THE INVENTION

Despite the recent discovery of insulin and its widespread use in the treatment of diabetes, and despite the later discovery and use of sulfonylureas / such as Chlorpropamide ( ^R ) (Pfizer), Tolbutamide ( ^R ) (Upjohn), Acetohexamide ( ^R ) (EI Lilly), Tolazamide ( ^R ) (Upjohn)], biguanides (such as Phenformin ( ^R ) (Ciba Geigy), Metformin ( ^R ) (GD Searle)), alpha-glucosidase inhibitors (such as Precose ® ^R (Bayer)) and substances Insulin sensitivity enhancers / such as Rezulin ® ^R (Parke Davis), as oral hypoglycaemic agents, treatment of diabetes is still not sufficiently successful. Insulin, which is needed in approximately 10% of patients affected by diabetes in whom synthetic hypoglycaemic agents are not effective (type I diabetes, insulin dependent diabetes mellitus), should be administered several times a day, with injection usually being administered by the patient himself. Determining the correct insulin dosage requires frequent determination of blood sugar or urine. Administration of excess insulin causes hypoglycaemia, resulting in a wide range of abnormalities ranging from mild changes in blood glucose to coma or even death. Treatment of non-insulin dependent type diabetes mellitus (type II diabetes, NIDDM) usually involves a combination of diet, exercise, administration of oral agents such as sulfonylureas, and in more severe cases, insulin. However, clinically available hypoglycaemics may have side effects that limit their use. It is always true that if one of these agents fails in an individual case, others may be successful. Clearly, there remains a need for hypoglycaemic agents loaded with fewer side effects or showing efficacy when other hypoglycaemic agents fail.

Aldose reductase inhibitors constitute a class of compounds that are well known to be useful in the prevention and treatment of conditions caused by complications of diabetes, such as diabetic neuropathy and nephropathy. Such compounds are known to those skilled in the art and can be readily identified using standard bioassays.

Examples of compounds known to be useful as aldose reductase inhibitors include, but are not limited to,

3,4-dihydro-4-oxo-3- 5- (trifluoromethyl) -2-benzothiazolyl methyl-1-phthalazineacetic acid and a number of related compounds thereof, which are described in U.S. Patent 4,939,140 to Larson et al. This patent is incorporated herein by reference in its entirety. Comply with the structural formula

is an aldose reductase inhibitor and a compound useful in the treatment of the above complications induced by diabetes mellitus.

Certain aldose reductase inhibitors have been reported to be useful in lowering lipid levels in mammals - see, for example, U.S. Patent 4,492,706 (Kallai-sanfacon) and EP 0 310

931A2 (Etyl Corporation). These documents are incorporated herein by reference in their entirety.

US Patent 5,064,830 (Going) discloses the use of certain oxophthalazinylacetic acids, including zopolrestat, for lowering blood uric acid levels.

U.S. Pat. 08/059 688 discloses certain aldose reductase inhibitors, including zopolrestat, for lowering blood lipid levels in humans. This application discloses that therapeutic utility results from the treatment of diseases caused by elevated levels of triglycerides in the blood, such as cardiovascular disorders such as thrombosis, arteriosclerosis, myocardial infarction and angina pectoris.

Atherosclerosis, an arterial disease, is considered to be the leading cause of death in the US and Western Europe. The pathological sequence leading to atherosclerosis and occlusive heart disease is well known. At the most frequent stage of this sequence, fat strips are formed in the carotid, coronary and cerebral arteries as well as in the aorta. These lesions are yellow due to the presence of lipid deposits, which are found mainly in smooth muscle cells and in the macrophages of the intima layer of the artery and the aorta. It is believed that most of the cholesterol found in these fat strips results in fibrous coatings consisting of accumulated intimal smooth muscle cells folded by lipid and surrounded by extracellular lipid, collagen, elastin and proteoglycans. The cells form a fibrous cover with the matrix overlying deeper deposits of cell debris and other extracellular lipid. Lipid is predominantly free and esterified cholesterol. Fibrous coatings develop slowly and over time undergo calcification and necrotization, leading to complicated lesions resulting in occlusion of the artery and a tendency to mural thrombosis and arterial muscular spasm advanced arthrosis. .

Epidemiological evidence has firmly confirmed the hypothesis that hyperlipidemia is a major risk factor for cardiovascular disease (CVD) due to atherosclerosis. In recent years, leading physicians have put a new emphasis on lowering plasma cholesterol, especially low density lipoprotein cholesterol, as an important step in preventing CVD. It is known that the upper limit of the normal state is currently substantially lower than in the past. As a result, large sections of the Western world population are now considered particularly vulnerable. Such independent risk factors include glucose intolerance, left ventricular hypertrophy, hypertension, and male affiliation. Cardiovascular disease occurs in patients affected by diabetes, at least in part, because a greater number of independent risk factors are present in this part of the population. Successful treatment of hyperlipidemia in the whole population and especially in the population affected by diabetes is therefore of exceptional medical importance.

Hypertension (or high blood pressure) is a condition that occurs in the human population as a secondary symptom of various other disorders such as renal artery stenosis, peochromocytes, or endocrine disorders. However, hypertension also occurs in many patients whose causative agent or disorder is unknown. Essential hypertension is often associated with disorders such as obesity, diabetes and hypertriglyceridemia, but the relationship between these disorders has not yet been explained. In addition, many patients exhibit symptoms of high blood pressure, even though they otherwise have no other signs of disease or disorder.

It is known that hypertension can directly lead to heart failure, renal failure and stroke (cerebral hemorrhage). These can cause death in a short time. Hypertension may also contribute to the development of atherosclerosis and coronary disease. Such conditions gradually weaken the patient and may also result in death over an extended period of time.

The exact cause of essential hypertension is unknown, although a number of factors are believed to initiate the disease. These factors include stress, uncontrolled emotions, unregulated hormone release (renin, angiotensin, aldosterone system), excessive salt and water retention due to poor kidney function, wall thickening and vasculature hypertrophy leading to vascular constriction, as well as genetic factors.

Treatment of essential hypertension is approached by taking all these factors into account. Thus, a wide variety of blockers, vasoconstrictors, angiotensin converting enzyme inhibitors and the like have been developed and marketed as antihypertensive agents. The treatment of hypertension using these compounds has been shown to be beneficial in preventing short-term death, as well as preventing heart failure, gastrointestinal failure, and cerebral hemorrhage. However, the problem of gradual long-term development of atherosclerosis or heart disease due to hypertension remains. Thus, although high blood pressure is reduced, the underlying cause of essential hypertension does not respond to such treatment.

Hypertension has been associated with an increased level of insulin in the blood, a condition called hyperinsulinemia. Insulin is a peptide hormone whose main effect is to ensure glucose utilization, protein synthesis and neutral lipid production and storage. Among other things, insulin also promotes vascular cell growth and increases sodium retention in the kidneys. Finally, these functions can be achieved without affecting glucose levels and are known causes of hypertension. For example, the growth of peripheral vasculature may result in the constriction of peripheral capillaries and sodium retention increases blood volume. Thus, a decrease in insulin level may prevent abnormal vascular growth and renal sodium retention due to high insulin levels in hyperinsulinemics, thereby reducing hypertension.

Cardiac atrophy is a significant risk factor leading to sudden death, myocardial infarction and partial heart failure. These cardiac events are at least partly due to increased susceptibility to myocardial damage following ischemia and reperfusion, and may occur as in outpatients.

·· • · • · ·· • ··· ·· • · 9 · • • • • · · 9 · • · • • · • • · • · · · • · • · · · • · · • · · · ···· • • • · · • · • · • ···

patients and perioperative cases. Attempts to prevent or minimize the adverse perioperative effects on the myocardium, in particular to prevent or minimize the perioperative myocardial infarction, have not been fully successful. Both non-cardiac and cardiac surgery are associated with a substantial risk of myocardial infarction or death. About 7 million patients undergoing non-cardiac surgery are expected to be at risk, with the incidence of perioperative death and severe cardiac complications up to 20-25% in some types of operations. In addition, about 400,000 patients undergo coronary bypass surgery each year, and in this population the incidence of perioperative myocardial infarction is estimated to be 5% and the number of deaths is 1-2%. Currently, there is no pharmacotherapy available to reduce cardiac tissue damage to perioperative myocardial ischemia or to increase cardiac resistance to ischemic episodes. It is believed that such therapy could save human lives and reduce hospitalization, increase the quality of life and reduce the overall cost of healthcare for high-risk patients.

The production of glucose in the liver is an element that is important in the focus of therapy

NIDDM.

The liver is a major regulator of plasma glucose levels in the post-absorbent state (fasting) and the rate of hepatic glucose production in NIDDM patients is significantly higher compared to the normal population. Similarly, in the postprandinal (post-meal) state, where the liver has a proportionally lesser effect on total plasma glucose, liver glucose production is abnormally high in NIDDM patients.

Glycogenolysis is an important target that is being sought to stop hepatic glucose production. Liver produces

• t • · · · • • · • · • • ·· • ·  • · • · · · · • · • · • · • · • · · · • · • · · · • · · • · • · ···· • • • · · • · · · • ···

glucose by glycogenolysis (by decomposing glycogen, which is a glucose polymer) and by gluconeogenesis (by synthesis of glucose from 2- and 3-carbon precursors). Several series of evidence show that glycogenolysis can make an important contribution to liver glucose production in the treatment of NIDDM. First of all, a normal postabsorptive human is estimated to produce about 75% of the glucose in the liver by glycogenolysis. In addition, patients afflicted with diseases that impair hepatic glycogen storage, such as those suffering from Hers disease (glycogen phosphorylase deficiency), show episodes of hypoglycemia. These observations support the assumption that glycogenolysis may be an important way of producing glucose in the liver.

Glycogenolysis is catalyzed by the liver, muscle and brain by tissue-specific isoforms of the enzyme glycogen phosphorylase. This enzyme cleaves the glycogen macromolecule by releasing glucose-1-phosphate and a new truncated gycogen macromolecule. To date, two types of glycogen phosphorylase inhibitors have been reported: glucose and glucose analogs (Martin JL et al., Biochemistry, 1991, 30, 10101) and caffeine and other purine analogs (Kasvinsky PJ et. Al., J.Biol.Chem. 1978, 253, 3343-3351 and 9102-9106). These compounds and glycogen phosphorylase inhibitors are generally considered to be potentially useful in the treatment of NIDDM by reducing hepatic glucose production and reducing blood glucose (Blundell,

T. B. et al., Diabetology 1992, 35. Suppl. 2, 569-576 and Martin et al., Biochemistry 1991, 30, 10101).

The mechanism or mechanisms responsible for myocardial damage observed in ischemia and reperfusion are not fully explained. It has been reported (MF Allard et al., Am. J. Physiol. 267, H66-H74, 1994) that preischemic reduction of glycogen ... is associated with improved postischemic functionalities. · ········································ · · · · · · · · · · · · · · Left ventricular recovery> in hypertrophized rat heart.

Thus, although there are a variety of treatments for hyperglycemia, hypercholesterolemia, hypertension, hyperinsulinemia, atherosclerosis hyperlipidemia, and ischemia, efforts remain to develop alternative treatments.

SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising an aldose reductase inhibitor and a glycogen phosphorylase inhibitor. The invention further provides the use of a composition for the treatment of insulin resistance conditions such as diabetes in mammals (e.g., humans, women or men) and use to reduce tissue damage (e.g., substantially preventing tumor damage, inducing tissue protection) induced by ischemia.

The combination comprises therapeutically effective amounts of an aldose reductase inhibitor and a glycogen phosphorylase inhibitor.

A preferred amount of aldose reductase inhibitor is about 0.1 mg / kg to about 20 mg / kg and a preferred amount of glycogen phosphorylase inhibitor is about 0.1 mg / kg to about 15 mg / kg.

Particularly preferred as an aldose reductase inhibitor is 3,4-dihydro-4-oxo-3- [5- (trifluoromethyl) -2-benzothiazolyl] methyl] -1-phthalazineacetic acid.

Preferred glycogen phosphorylase inhibitors include compounds of Formula I

· · · · · · • • • • • · • • • · · · • · • · • • • · • · • ···· e • • · • • • · · • · · * •

• · · ·

the dotted line represents an optional bond, represents a group of formula -C (H) =

C (alkyl) = C 1 -C 4 alkyl or -C (halogen) = when the dashed line represents a bond, or A is a methylene group or a group of the formula -CH (alkyl) - C 1 -C 4 alkyl parts where the dashed line does not represent a bond,

R _{1 O} and R _1Z rji are each independently H, halo, 4-, 6-or 7-nitro, cyano, C 1 -C 4 alkyl, C 1 -C 4, C atoms, fluoromethyl, difluoromethyl or trifluoromethyl,

R2 represents a hydrogen atom,

R ₃ is hydrogen or C 1 -C 5 alkyl,

R ₄ is hydrogen, methyl, ethyl, n-propyl, C 1 -C 3 hydroxyalkyl, C 1 -C 3 alkoxyalkyl and C 1 -C 3 alkyl, C 1 -C 4 phenylalkyl C1-C4-phenyl-hydroxyalkyl, phenyl-C1-C4-alkoxy and C1-C4-alkyl, thiene-2- or -3-yl-C1-C4-alkyl; or a (C 2 -C 4) -alkyl or C 1-4 alkyl group wherein the ring in R 4 is optionally substituted on carbon by one, two or three substituents independently selected from the group consisting of hydrogen , a halogen atom of a C1-C4 alkyl group, a C1-C4 alkoxy group, a trifluoromethyl group, a hydroxy group, an amino group and a cyano group or

R ₄ is pyrid-2-, -3- or -4-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety, thiazol-2-, -4- or -5-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety, imidazole -1-, -2-, -4- or -5-ylalkyl having 1 to 4 carbon atoms in the alkyl moiety, pyrrol-2- or -3-ylalkyl having 1 to 4 carbon atoms in the alkyl moiety, oxazol-2-, -4- or -5-ylalkyl having 1 to 4 carbon atoms in the alkyl moiety, pyrazol-3-, -4- or -5-ylalkyl having 1 to 4 carbon atoms in the alkyl moiety, isoxazole-3-, -4- or C 1 -C 4 -alkyl, C 1 -C 4 -alkyl, isothiazol-3-, -4- or -5-C 1 -C 4 -alkyl, pyridazine-3- or -4-C 1 -C 4 -alkyl; carbon atoms in the alkyl moiety, pyrimidine-2-, -4-, -5- or

(C 1 -C 4) -alkyl, (C 1 -C 4) -alkyl, (C 1 -C 4) -pyrazin-2- or (3-ylalkyl) or (C 1 -C 4) -benzyl-2-ylalkyl; in the alkyl moiety wherein the heterocyclic ring contained in R 4 is optionally substituted on carbon by one or two substituents independently selected from the group consisting of halogen, trifluoromethyl, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, amino and hydroxy groups.

· · • e · · • ·· • · • • ·· • · • ·· 9 • · · · • · • · • • * • • · · • · · · «· • · • * • · ·· • · ·· • · ·· • • • · · · • ···

is hydrogen, hydroxy, fluoro, (C1-C6) alkyl, (C1-C6) alkoxy, (C1-C6) alkanoyl, (C1-C4) aminoalkoxy, mono-N- or di-N, N -alkylaminoalkoxy of 1 to 4 carbon atoms in each of the alkyl and 1 to 4 carbon atoms in each of the alkoxy moieties, carboxyalkoxy of 1 to 4 carbon atoms in the alkoxy moiety, alkoxycarbonylalkoxy of 1 to 5 carbon atoms in the first and 1 to 4 carbon atoms in the second alkoxy moiety, a C 1 -C 4 benzyloxycarbonylalkoxy or carbonyloxy moiety, which is attached via a carbon-carbon bond to the phenyl, thiazolyl, imidazolyl, indo-indolyl, furyl, oxyl, oxyl, oxyl, pyridazinyl, pyrimidinyl, or 1,3,5-triazinyl ring isotiazo wherein the ring in R ₅ is optionally substituted on carbon by one substituent selected from the group consisting of halogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, hydroxy, amino and trifluoromethyl represents a hydrogen atom, a fluorine atom or an alkyl group having from 1 to 5 carbon atoms;

R ₅ R ₇ together optionally represent an oxo group,

R ₆ is carboxy, C 1 -C 8 alkoxycarbonyl or C (O) NR 8 R 8 or C (O) R ₁₂ wherein:

and · · · · • · · • • • • • • • • in • · • • · · · • e • · • • • and • · • · • ···· • · * * • · ·· • · ·· • · ·· • • • · ·· • · · ·

R 8 is hydrogen, alkyl of 1 to 3 carbon atoms, hydroxy or alkoxy of 1 to 3 carbon atoms, and

R ₉ is hydrogen, C 1 -C 8 alkyl, hydroxy, C 1 -C 8 alkoxy, C 1 -C 8 methylene-perfluorinated alkyl, phenyl, pyridyl, thienyl, furyl, pyrrolidinyl, pyrrolidinyl , oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, pyranyl, piperidyl, morpholinyl, pyridazinyl, pyridazinyl, pyrimidinyl, pyrimidinyl, pyrimidinyl, pyrimidinyl carbon-nitrogen bonds; or

R 8 is C 1 -C 5 alkyl substituted with one, two or three substituents independently selected from the group consisting of hydrogen, hydroxy, amino, mono-N- or di-N, N-alkylamino of 1 to 5 atoms carbon in each of the alkyl moieties; or

R 8 is C 1 -C 5 alkyl substituted with one or two substituents independently selected from the group consisting of phenyl, pyridyl, furyl, pyrrolyl, pyrrolidinyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazol, pyranyl, pyridyl, piperidyl, morpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl,

• · · · · · • · · • and • • · • • • • · • • · · · • • • · • • * and and • · • · • ···· • · • 9 • · ·· • · ·· • · ·· • • • · ·· β ···

piperazinyl and 1,3,5-triazinyl, wherein the non-aromatic nitrogen-containing ring of R _g is optionally substituted on the nitrogen atom with one C 1 -C 6 alkyl, benzoyl benzoyl or C 1 -C 6 alkoxycarbonyl alkoxycarbonyl and wherein the ring located at the R _g is optionally substituted on carbon by one substituent selected from the group consisting of halogen, C 1 -C 4 alkyl, C 1 -C 4 alkyl, hydroxy, amino, and mono-N- and di-N, N-C 1 -C 5 -alkylamino groups in each alkyl moiety, provided that it contains no quaternized nitrogen atom and no nitrogen-oxygen, nitrogen-nitrogen or nitrogen-halogen bond,

R ₁₂ is piperazin-1-yl, 4-alkylpiperazin-1-yl of 1 to 4 carbon atoms in the alkyl moiety, 4-formylpiperazin-1-yl, morpholino, thiomorpholino, 1-oxothiomorpholino,

1,1-dioxothiomorpholino, thiazolidin-3-yl, 1-oxothiazolidin-3-yl, 1,1-dioxothiazolidin-3-yl, 2-alkoxycarbonylpyrrolidin-1-yl of 1 to 6 carbon atoms in the alkoxy moiety, oxazolidin-3 -yl or 2 (R) -hydroxymethylpyrrolidin-1-yl, or

R ₂ is 3- and / or 4-mono or -disubstituted oxazetidin-2-yl, 2-, 4- and / or 5-mono or -disubstituted oxazolidin-3-yl, 2-, 4-, and / or 5 - mono- or -disubstituted thiazolidine-314

the · · · · • · · • • • • · • • · · • • · · · • · • · • · and • · • · · ···· • · · • · ·· • · ·· • · ·· • • • · ·· ·

2-, 4-, and / or 5-mono- or di-substituted 1-oxothiazolidin-3-yl, 2-, 4-, and / or 5-mono- or di-substituted 1,1-dioxothiazolidine 3-yl, 3- and / or 4-mono- or -disubstituted pyrrolidin-1-yl, 3-, 4-, and / or 5-mono-, di- or -trisubstituted piperidin-1-yl, 3- 4- and / or 5-mono-, di- or -trisubstituted piperazin-1-yl, 3-substituted azetidin-1-yl, 4- and / or 5-mono- or di-substituted 1,2-oxazinan-2-yl, 3- and / or 4-mono- or -disubstituted pyrazolidin-1-yl, 4- and / or 5-mono- or disubstituted isoxazolidin-2-yl, 4- and / or 5-mono- or -disubstituted isothiazolidin-2 each of the substituents independently selected from the group consisting of hydrogen, halogen, alkyl of 1 to 5 carbon atoms, hydroxy, amino, mono-N- and di-N, N-alkylamino of 1 to 5 atoms carbon in each of the alkyl moieties, formyl pins, oxo, hydroxyimino, C 1 -C 5 alkoxy, carboxy, carbamoyl, mono-N- and di-N, N-C 1 -C 4 alkylcarbamoyl in each of the alkyl moieties, C 1 -C 4 alkoxy, (C 1 -C 4) alkoxymethoxy, (C 1 -C 6) alkoxycarbonyl, (C 1 -C 5) carboxyalkyl and (C 1 -C 5) hydroxyalkyl group, provided, however, that: • ······················································ If R ₄ is hydrogen, methyl, ethyl or n-propyl, then R 8 is hydroxy when R ₅ and R ₇ are hydrogen; are hydrogen, then R _{4 is} not hydrogen, methyl, ethyl, n-propyl, C 1 -C 3 hydroxyalkyl or C 1 -C 3 alkoxyalkyl and 1 to 3 carbon atoms in the alkyl moiety, and R _g is C (O) NR _g R _g or C (O) R ₁₂ or C 1 -C 4 alkoxycarbonyl, and pharmaceutically acceptable salts and prodrugs thereof.

A first group of preferred compounds are those compounds of formula I wherein

R 1 is 5-H, 5-halogen, 5-methyl or

5-cyano, ^{R10 and} Ru each represent a hydrogen atom or a halogen atom, represents a group of the formula -C (H) =,

R ₂ and R ₃ are H;

represents a phenylalkyl group having 1 to 2 carbon atoms in the alkyl moiety, wherein said phenyl group is substituted with one, two or three substituents independently selected from the group consisting of hydrogen and halogen, or one or two substituents independently selected from the group consisting of hydrogen. halogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, tri16

• a · · · · • · · • • • • • · • • * • · • • · · · • · • · • • • Λ • • · • · · ···· • · • A • · ·· • · ·· • · ·· • • • · · · * ···

fluoromethylscins, hydroxy, amino and cyano; or

R ₄ is thien-2- or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, pyrid-2-, -3 or -4-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, thiazol-2-, -4- or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, imidazole-1-, -2-, -4- or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, fur-2- or -3- C 1 -C 2 alkyl, pyrrol-2- or -3-C 1 -C 2 alkyl, oxazol-2-, -4- or -5-C 1 -C 2 alkyl alkyl, pyrazol-3-, -4- or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, isoxazol-3-, -4- or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, wherein: the heterocyclic radical contained in R ₄ is optionally substituted on carbon by one or two substituents independently selected from the group consisting of halogen, trifluoromethyl, alkyl C1 -C4 -alkoxy, C1 -C4 -alkoxy, amino and hydroxy are hydroxy;

^R 6 is C (O) NR ₈ R ₉ or C (O) R ₁₂ , a

R?

represents a hydrogen atom.

Of the aforementioned first group of preferred compounds, particular preference is given to compounds falling within the first group of especially preferred compounds of formula I, wherein:

atom carbon and has (S) stereochemistry, atom carbon b has (R) stereochemistry, ^R 4 is phenyl having 1 to 2 carbon atoms in the alkyl moiety, thien-2-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, thien-3-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, fur-2-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, or fur-3ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, each of the above rings being substituted with one or two substituents independently selected from the group consisting of hydrogen and fluorine, ^R 6 It is C (O) NRgR _g. ^R 8 is (C1-C3) -alkyl, hydroxy or (C1-C3) -alkoxy; and rg is hydrogen, alkyl of 1 to 8 carbon atoms, hydroxy, hydroxyalkyl of 1 to 6 carbon atoms, alkyloxy of 1 to 8 carbon atoms, pyridyl, morpholinyl, piperazinyl, pyrrolidinyl, piperidyl, imidazolyl or thiazolyl or (C 1 -C 4) alkyl substituted with one substituent selected from the group consisting of pyrrolidinyl, piperidyl, imidazolyl or thiazolyl;

morpholinyl, piperazinyl, pyrrolidinyl, piperidyl, imidazolyl and thiazolyl groups.

································································· I · ·· ···

Of the first group particularly preferred, it favors specific compounds selected by the following: compounds from file

5-Chloro-1H-indole-2-carboxylic acid 5 (chloro) - ((R) -hydroxydimethylcarbamoylmethyl) -2-phenylethyl] -amide; R (1S) - (R) -hydroxy (methoxymethylcarbamoyl) methyl] -2- 5,6-Chloro-1H-indole-2- (5,6-dichloro-1H-indole-2-carboxylic acid phenylethyl) amide, [(1S) -? (R) -hydroxy (methoxymethylcarbamoyl) methyl] -2-phenylethyl] amide 5-chloro-1H-indole-2-carboxylic acid ((S) - (R) -hydroxy- (2-hydroxyethyl) methylcarbamoyl methyl 2-phenylethyl) amide,

5-Chloro-1H-indole-2-carboxylic acid methyl-2-phenylethyl-amide and ((S) - {(R) -hydroxy- [(methyl) - (R) -hydroxy (methylpyridin-2-ylcarbamoyl) methyl-2-phenylethyl 5-Chloro-1H-indole-2-carboxylic acid (2-pyridin-2-yl-ethyl) -carbamoyl-methyl-2-phenylethyl) -amide.

Of the first group of particularly preferred compounds, compounds of formula I are preferred, wherein

a. R is 5-chloro, R _10, and R _{i, l} are H; _R4 is benzyl; _R8 is methyl and R is methyl, ·· ·· ·· ·· · · · · · · • • ··· · ··· · · · · · · · · · · · · · · · · · · · · · · ·

b. R is 5-chloro, Rpredstavuje H, R ₀ is 6-chloro; _R4 is benzyl, R is methyl and R ₉ is methoxy,

c. R ₁ is 5-chloro, R @ ₁₀ and R @ _{1, L} is H, R ₄ is benzyl, R _g is methyl and _Rg is methoxy.

d. R is 5-chloro, R @ ₁₀ and R @ _{1, L} is H, R ₄ is benzyl, R _g is methyl, and R ₉ is 2- (hydroxy) -etylskupinu.

e. R 1 is 5-chloro, R ₁₀ and R ₁₁ are hydrogen, R ₄ is benzyl, R 8 is methyl and R ₉ is pyridin-2-yl; and

f. Rl is 5-chloro, R _10, and R _{i, L} is H, R ₄ is benenzylskupinu R _g is methyl and _Rg is 2- (pyridin-2-yl) ethyl.

Of the aforementioned first group of preferred compounds, the compounds belonging to the second group of particularly preferred compounds of formula I are also more preferred, wherein:

atom carbon and has (S) stereochemistry, atom carbon b has (R) stereochemistry, ^R 4 is phenylalkyl having 1 to 2 carbon atoms in the alkyl moiety, a thien-2-ylalkyl of 1 to 2

································ Carbon atoms in the alkyl moiety, thien-3-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, fur-2-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, or fur- 3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, each of the above rings being substituted with one or two substituents independently selected from the group consisting of an atom independently selected from hydrogen and fluoro, ^R 6 is C (O) R ₁₂ , ^r 12

4-alkylpiperazin-1 is yl

3-substituted azetidin-1-yl, mono- or -disubstituted nu, 4- and / or 5-mono- or xazolidin-2-yl, -disubstituted 1,2-oxazinan-2-yl, each of the substituents of the group consisting of The group consisting of halogen, hydroxy, di-NN-alkylamino having 1 to 6 carbon atoms in each of the alkyl moieties, oxo, hydroxyimino and alkoxy.

morpholino having up to 4 carbon atoms in the alkyl moiety, 3- and / or 4-pyrrolidin-1-yl-disubstituted iso4- and / or 5-mono- or independently selected from hydrogen, amino, mono-N- and above said second group separately

Of the preferred compounds, a compound selected from the group consisting of:

5-Chloro-1H-indole-2-carboxylic acid (E) - (1S) -benzyl- (2R) -hydroxy-3- (4-methyl-piperazin-1-yl) -3-oxo-propyl] -amide,

5-Chloro-1H-indole-2-carboxylic acid C (1S) -benzyl- (2R) -hydroxy-3- (3-hydroxy-azetidin-1-yl) -3-oxopropyl-amide, ·· ·· ··· ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 5-chloro-1H-indole-2-carboxylic acid ((1S) -benzyl- (2R) -hydroxy-3-isoxazolidin-2-yl-3-oxopropyl) -amide, ((1S) -benzyl- (2R)) 5-Chloro-1H-indole-2-carboxylic acid-hydroxy-3- (1,2-oxazinan-2-yl-3-oxopropyl) -amide, N (1S) -benzyl- (2R) -hydroxy-3 - ((3S) ) -hydroxy-pyrrolidine-l-yl) -

5-Chloro-1H-indole-2-carboxylic acid 3-oxopropyl-7, [(1S) -benzyl-3 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl] -amide 5 - chloro-1H-indole-2-carboxylic acid,

5-Chloro-1H-indole-2-carboxylic acid pS) -benzyl-3 - ((3R, 4S) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide, ((1S) -benzyl) 5-Chloro-1H-indole-2-carboxylic acid (2R) -hydroxy-3-morpholin-4-yl-3-oxopropyl) -amide.

A second group of particularly preferred compounds provides a compound of formula I wherein:

a. R 1 is 5-chloro, R ₁₀ is hydrogen, R ₄ is benzyl

4-methylpiperazin-1-yl,

b. Rl is 5-chloro, R _10, and R _{i, l} are H; _R4 is benzyl and R ₁₂ is

3-Hydroxy-l-yl,

c. R 5 is 5-chloro, R ₁₀ and R _{11; L} are hydrogen, R ₄ is benzyl, and R ₁₂ is isoxazolidin-2-yl, R ₄ is benzyl and R ₁₂ is isoxazolidin-2-yl; ··· e · · · · · · · · · · · · · · · · · · · · · ·

d. R 1 is 5-chloro, R ₁₀ and R 6 are hydrogen, R ₄ is benzyl and R ₁₂ is (1,2) -oxazinan-2-yl,

e. R ₁ is 5-chloro, R ₁₀ and R _{1; L} are hydrogen, R ₄ is benzyl and R ₁₂ is 3 (S) -hydroxypyrrolidin-1-yl,

f. R is 5-chloro, R _10, and R _{i, l} are H; _R4 is benzyl and R ₁₂ is (3S, 4S) -dihydroxy-l-yl,

g. is 5-chloro, R _10, and R _{i, l} are H; _R4 is benzyl and R ₁₂ is (3S, 4S) -dihydroxy-l-yl, and

h. Rl is 5-chloro, R _10, and R _{i, l} are H; _R4 is benzyl and R ₁₂ is morpholino.

A second group of preferred compounds are those compounds of formula (I) wherein hydrogen, halogen, methyl or cyano, ^R 10 ^{and R} 11 are each independently hydrogen or halogen,

A is -C (H) =,

R ₂ and R ₃ are H;

R ₄ is phenylalkyl having 1 to 2 carbon atoms in the alkyl moiety, said phenyl group 23

· · · · • · • · · • • • • • • • • · • · • • · · · • • • · • · • • • • · • · • ···· • · ♦ • • · · · · · · • · · • · ·

na is substituted with one, two or three substituents independently selected from the group consisting of hydrogen and halogen, or one or two substituents independently selected from the group consisting of hydrogen, halogen, C 1 -C 4 alkyl, alkoxy (C 1 -C 4), trifluoromethyl, hydroxy, amino or cyano, or

is thien-2- or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, pyrid-2-, -3- or -4-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, thiazol-2-, - 4 or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, imidazole-1-, -2-, -4- or

-5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, fur-2- or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, pyrrol-2- or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety oxazole-2-, -4- or -5-alkyl of 1 to 2 carbon atoms in the alkyl moiety, pyrazol-3-, -4- or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, isoxazol-3- 5-ylalkyl having 1 wherein R ₄ is one

-4- or carbon in the alkyl moiety optionally substituted by substituents independently selected from the group consisting of a T atom, an alkyl group having 1 to amino and hydroxy groups having up to 2 heterocyclic atoms on carbon or two of the halogen atom, up to 4 carbon atoms, carbon atoms,

R ₅ is hydroxy,

· · · · · · • · · • • • • • • • • · · · • • · · · • • • · • · • • • • · • · • ···· · • · • • · · · · · · • · · · · ·

R 8 is carboxy or C 1 -C 8 alkoxycarbonyl in the alkoxy moiety f and

R ₇ is a hydrogen atom, a fluorine atom or an alkyl group having 1 to 6 carbon atoms.

Of the aforementioned second group of preferred compounds, more preferred compounds of formula I are those wherein carbon atom a has (S) stereochemistry, carbon atom b has (R) stereochemistry,

R ₄ is phenylalkyl having 1 to 2 carbon atoms in the alkyl moiety, thien-2-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, thien-3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, fur-2-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety 1 to 2 carbon atoms in the alkyl or fur-3-yl C 1 -C 2 alkyl, each of the above rings are substituted with one or two substituents independently selected from the group consisting of H or F, ^R 10 11 are hydrogen atoms,

R ₆ is carboxy; and R ₇ is hydrogen.

Preferred from the immediately preceding group are compounds of formula I wherein

R-L is 5-chloro,

· · · · · · • • · • • • • • • • · · • • · · · • • • · • · • • • · • · • ···· • · · • · · · · · · • ·· ·

^R 10 ^{and R} 11 P ^re dstavujú hydrogen atoms and benzyl. ^R 4 is Third group preferred compounds form compound of of formula I wherein

^{R @} 10 ^{and R @} 11 are each independently hydrogen or halogen, ^{R @} 10 ^{and R @} 11 are each independently hydrogen, halogen, methyl or cyano;

A is -C (H) =,

R ₂ and R ₃ are H;

R ₄ is phenylalkyl having 1 to 2 carbon atoms in the alkyl moiety, wherein said phenyl is substituted with one, two or three substituents independently selected from the group consisting of hydrogen, halogen or one or two substituents independently selected from the group consisting of hydrogen, halogen, C1-C4alkyl, C1-C4alkoxy, trifluoromethyl, hydroxy, amino and cyano, or

R ₄ is thien-2- or -3-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, pyrid-2-, -3- or -4-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, thiazol-2-, -4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, imidazlol-1-, -2-, -4- or -5-ylalkyl 26 1 to 2 carbon atoms in the alkyl moiety, fur-2- or -3-ylalkyl of 1 to 2 atoms carbon in the alkyl moiety, pyrrol-2- or -3-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, oxazol-2-, -4- or -5-yl-alkyl of 1 to 2 carbon atoms in the alkyl moiety, pyrazole -3-, -4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, isoxazole-3-, -4- or -5ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, the heterocyclic moiety being found in R ₄ is optionally substituted on carbon by one α1 or two substituents independently selected from the group consisting of halogen, trifluoromethyl, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, amino, and hydroxy,

R ₅ is fluorine, C 1 -C 4 alkyl, C 1 -C 5 alkoxy, C 1 -C 4 aminoalkoxy, C 1 -C 4 mono-N- or di-NN-alkylaminoalkoxy alkoxy and from 1 to 4 carbon atoms in each of the alkoxy moieties, from 1 to 4 carboxyalkoxy in the alkoxy moiety, from 1 to 5 carbon atoms in the first and 1 to 4 carbon atoms in the second alkoxy moiety or from 1 to 4 benzyloxycarbonylalkoxy in the alkoxy moiety, r ₆ is a carboxy or C 1 -C 8 alkoxycarbonyl moiety, and

R ₇ is a hydrogen atom, a fluorine atom or an alkyl group having 1 to 6 carbon atoms.

··················································· · · · · · · · ·

The fourth compounds ^R 1 ^R 10 ^{and R} 11 ^R 2 ^{and R} 3 ^R 4 ^R 4 are a group of preferred formulas I wherein the compounds form a hydrogen atom, a halogen atom, a methyl group or a cyano group each independently represents a halogen atom, a hydrogen atom or represents a group of the formula hydrogen atoms having 1 to 2 atoms wherein said phenylalkyl group of the alkyl moiety is substituted with one, two or a group, halogen independently represents carbon in the phenyl group with three substituents independently selected from the group consisting of hydrogen and atom or one or two substituents selected from the group consisting of is selected from hydrogen, halogen, C1-C4alkyl, C1-C4alkoxy, trifluoromethyl, hydroxy, amino and cyano, or is thiene-2- or -3-ylalkyl of 1 to 2 carbon atoms an alkyl-3- or -4-ylalkyl group having 1 in the alkyl group; parts, thiazole-2-, alkyl of 1 to 2 part atoms, pin of fur-2-carbon part of alkyl, pyrid-2-, up to 2 carbon atoms

-4- or -5-carbon in the alkyl or -5-ylalkyl moiety, with 3-ylimidazol-1-, -2-, -41 to 2 carbon atoms in the alkyl or -3-ylalkyl group having 1 to 2 in the alkyl moiety, pyrrole-2- or C 1 -C 2 alkyl

· · · · · · • · · • • • • • • • • • · · • • · · · • • • · • • • • • • • · • · • ···· • · • • · · · · · · • • · · · ·

oxazol-2-, -4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, pyrazol-3-, -4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, isoxazole- 3-, -4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, wherein the heterocyclic radical present in R ₄ is optionally substituted on carbon by one or two substituents independently selected from the group consisting of halogen, trifluoromethyl , (C 1 -C 4) alkyl, (C 1 -C 4) alkoxy, amino, and hydroxy groups,

R ₅ is fluorine, C 1 -C 4 alkyl, C 1 -C 5 alkoxy, C 1 -C 4 aminoalkoxy, mono-N- or di-N, N-C 1 -C 4 alkylaminoalkoxy C 1 -C 4 alkyl and C 1 -C 4 alkyl, C 1 -C 4 carboxyalkoxy, C 1 -C 5 alkoxy and C 1 -C 4 alkoxy, or C 1 -C 4 benzyloxycarbonylalkoxy the carbon atoms in the alkoxy moiety,

R ₆ is a group of formula C (O) NR _g R ₉ or C (O) R _12;

R ₇ is a hydrogen atom, a fluorine atom or an alkyl group having 1 to 6 carbon atoms.

Examples of preferred glycogen phosphorylase inhibitors include compounds of Formula IA

· · · · • · • · · • · • • • • • · · • • · · · • · • · • · • • • · · • · ···· • · · • • • · • • • • · · · · · · · • ·· ·

(ΙΑ) where the dashed line represents a possible bond,

A is -C (H) =,

-C (alkyl) = C 1 -C 4 alkyl, -C (halogen) = or -N = when the dotted line represents a bond, or A is a methylene group or a group of the formula -CH (alkyl) - with 1 to 4 C4 alkyl, where the dotted line is not a bond, ^R l ^{'and R} 10 ^R 11 p ^{re °} tavuje each independently hydrogen, halogen, cyano, 4-, 6- or 7-nitro, C 1 -C C 4 -C 4 alkoxy, C 1 -C 4 alkoxy, fluoro methyl, difluoromethyl or trifluoromethyl,

R ₂ represents

R ₃ represents a -C 5 H, H or C atoms,

R ₄ is hydrogen, methyl, ethyl, n-propyl, hydroxy (C 1 -C 3) alkyl, (C 1 -C 3) alkoxyalkyl

· · · · · · • ·· · • • • • • • • · · · • • · · · • • • · • · • • · • · • · • ···· · • · • • • • · • • • • · • · · · · · · • · · · · ·

alkoxy and 1 to 4 carbon atoms in the alkyl moiety, phenylalkyl of 1 to 4 carbon atoms in the alkyl moiety, phenylhydroxyalkyl of 1 to 4 carbon atoms in the alkyl moiety, phenylalkoxyalkyl of 1 to 4 carbon atoms in the alkoxy and 1 to 4 carbon atoms in the alkyl moiety, a thien-2- or -3-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety or a fur-2- or -3-ylalkyl group of 1 to 4 carbon atoms in the alkyl moiety, the ring present in ₄ is substituted on carbon by one, two or three substituents independently selected from the group consisting of hydrogen, halogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, trifluoromethyl, hydroxy, amino, cyano and 4, Or 5-dihydro-1H-imidazol-2-yl;

is pyridin-2-, -3- or -4-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety, thiazol-2-, -4- or -5-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety, imidazol-2 -, -4- or -5-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety, pyrrol-2- or -3-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety, oxazol-2-, -4 or -5-ylalkyl C 1-4 alkyl, pyrazol-3-, -4- or -5-ylalkyl C 1-4 alkyl, isoxazole-3-, -4- or -5-ylalkyl C 1-4 C1-C4-alkyl, thiazol-3-, -4- or -5-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety, pyridazin-3- or -4-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety, pyrimidine -2-, -4-, -5- or

C 1 -C 4 -alkyl-C 1 -C 4 -alkyl;

· · · · • · • · · • • • • • • • • • · · • • · · · • • • · • · • • • • · • · • ···· • · • • • • • · • · • • • • · · · · · · • • e • e ·

a keel, pyrazin-2- or -3-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety, 1,3,5 triazin-2-ylalkyl of 1 to 4 carbon atoms in the alkyl moiety or indol-2-alkyl of 1 to 4 carbon atoms Wherein the heterocyclic ring in R ₄ is optionally substituted on carbon by one or two substituents independently selected from the group consisting of halogen, trifluoromethyl, C 1 -C 4 alkyl, C 1 -C 4 alkyl up to 4 carbon atoms, amino, hydroxy and cyano;

R ₄ is R ₁₅ -carobnyloxymethyl, wherein R ₁₅ is phenyl, thiazolyl, imidazolyl, 1H-indolyl, furyl, pyrrolyl, oxazolyl, pyrazolyl, isoxazolyl, isothiazazolyl, pyridyl, pyridyl, pyridyl, pyridyl, pyridyl, pyridyl, pyridyl, pyridyl, pyridyl, pyridyl, pyridyl, pyridyl wherein the above R ₁₅ rings are optionally substituted on carbon by one or two substituents independently selected from the group consisting of halogen, amino, hydroxy, (C 1 -C 4) alkyl, (C 1 -C 4) alkoxy, and trifluoromethyl,

R ₅ represents a hydrogen atom,

R ₆ is carboxy, alkoxycarbonyl having up to 8 carbon atoms, alkoxycarbonyl, benzyloxycarbonyl or a group C (O) NR ₈ R _g, or -C (O) R _12, wherein

· · · · · · • · · • • • • • • • • • · · • · · · · • · • · • · • • • • · 4 · ···· • · • • • · • · • · • • · • · · · · · · • · · · · ·

R _g is H, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkyl in cykloalkyloej a C 1 -C 5 alkyl, hydroxy or C 1 -C 8 alkyl and ar ₉ is hydrogen, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkylalkyl and C 1 -C 5 cycloalkyl, C 4 -C 7 cycloalkenyl, C 3 -C 7 cycloalkylalkoxy and C 1 -C 5 alkoxy, C 3 -C 7 cyclalkyloxy, hydroxy, C 1 -C 8 methylene-perfluorinated alkyl, phenyl, or a heterocyclic group selected from the group consisting of pyridyl, furyl, pyrrole, pyrrole, pyrrole, pyrrole, pyrrole , pyrrolidinyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl yl, pyrazolinyl, pyrazolidinyl dinylskupiny, isoxazolyl, izotiazolylskupiny, pyranyl, pyridinyl, piperidinyl, morpholinyl, guanidinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl, benzothiazolyl, benzimidazolyl benzoxazolyl, tiochromanylskupiny and tetrahydrobenzotiazolylskupiny, wherein the group is heteroyklická attached through a carbon-nitrogen bond; or

R ₉ is C 1 -C 6 alkyl or C 1 -C 8 alkoxy, each optionally substituted with one substituent selected from the group consisting of C 4 -C 7 cycloalken-1-yl, phenyl thienyl, pyridyl, furyl, pyrrolyl, pyrrolidinyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl,

1,1-dioxothiomorpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl and indosilyl groups and each of the above C 1 -C 6 alkyl and C 1 -C 8 alkoxy is optionally additionally substituted with one or more two substituents independently selected from the group consisting of halogen, hydroxy, C 1 -C 5 alkoxy, amino, mono-N- and di-N, C 1 -C 5 -alkylamino in each of the alkyl moieties, cyano , carboxy and alkoxycarbonyl having 1 to 4 carbon atoms, alkoxy, and wherein any of the rings in R _g is alkyl optionally substituted by one or two substituents independently selected from the group consisting of a halogen atom, C 1 -C 4 alkyl, C C 1-4 alkyl, hydroxy, C 1-4 hydroxyalkyl and, (C 1 -C 4) aminoalkyl, (C 1 -C 4) mono-N- and di-N, N-alkylamino (C 1 -C 4) alkyl, (C 1 -C 4) alkoxyalkyl and (C 1 -C 4) alkoxyalkyl; alkyl moieties, amino groups, mono-N- and di-N, N-alkylamino34 < tb >< tb >< tb > C 1 -C 4 carbon atoms in each of the alkyl moieties, cyano, and C 1 -C 4 groups , carboxy, C 1 -C 5 alkoxycarbonyl, carbamoyl, formyl and trifluoromethyl, and each R _g is optionally substituted with one or more substituents independently selected from the group consisting of C 1 -C 5 alkyl and halogen with the proviso that none of the heterocycles in R _g is quartenized nitrogen atom,

R ₁₂ is morpholino, thiomorpholino,

1-oxothiomorpholino, 1,1-dioxothiomorpholino, thiazolidin-3-yl, 1-oxothiazolidin-3-yl, 1,1-dioxothiazolidin-3-yl, pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl, piperazin-4-yl, azetidin-1-yl, 1,2-oxazinan-2-yl, pyrazolidin-1-yl, isoxazolidin-2-yl, isothiazolidin-

2-yl, 1,2-oxazetidin-2-yl, oxazolidin-3-yl, 3,4-dihydroisoquinolin-2-yl, 1,3-dihydroisoindol-2-yl, 3,4-dihydro-2H-quinol- 1-yl, 2,3-dihydrobenzo [1,4] oxazin-4-yl, 2,3-dihydrobenzo [1,4] thiazine-

4-yl, 3,4-dihydro-2H-quinoxalin-1-yl,

3,4-dihydrobenzo [cis] - [1,2-oxazin-1-yl], 1,4-dihydrobenzo [d] [1,2-oxazin-3-yl], 3,4-dihydrobenzo [eta] < 2 >, 2-oxazin-2-yl , 3H-benzo-dizoxazol-2-yl, 3H-benzo-cizoxazol-1-yl or azepan-1-yl, wherein each ring in R ₁₂ is optionally substituted with one, two or three substituents independently selected from the group consisting of C 1 -C 5 alkyl, C 1 -C 5 alkoxy, hydroxy, amino, mono-N- and di-N, N-alkylamino with up to 5 carbon atoms in each of the alkyl moieties, formyl , carboxy, carbamoyl, mono-N- and di-N, N-alkylcarbamoyl having 1 to 5 carbon atoms in each of the alkyl moieties, alkoxyalkoxy having 1 to 6 carbon atoms in the first and 1 to 3 carbon atoms in the other alkoxy moiety, (C 1 -C 5) alkoxycarbonyl, benzyloxycarbonyl, (C 1 -C 5) alkoxycarbonyl; C 5 alkoxy and 1 to 5 carbon atoms in the alkyl moiety, C 1 -C 4 alkoxycarbonylamino, 1 to 5 carboxy alkoxy moieties, carboxylalkyl C 1 -C 5 alkyl moieties, mono-N - and di-N, N-alkylcarbamoylalkyl of 1 to 5 carbon atoms in each of the alkyl moieties, hydroxyalkyl of 1 to 5 carbon atoms, alkoxyalkyl of 1 to 4 carbon atoms in the alkoxy and 1 to 4 carbon atoms in the alkyl moiety, aminoalkyl groups of C 1 -C 4, mono-N- and di-N, N-alkylaminoC 1 -C 4 alkyl in each of the alkyl moieties, oxo, hydroxyimino and C 1 -C 6 alkoxyimino, with no more than two substituents selected from the group consisting of oxo, hydroxyimino and C 1-6 alkoxyimino and oxo, hydroxyimino or alkoxyimino C 1-6 is attached to a non-aromatic carbon atom and wherein each R ₁₂ ring is additionally substituted with one or two substituents independently selected from the group consisting of C 1 -C 5 alkyl and halogen,

• e · · · · • · · • • • • • • • · • · • • · · · • • • · • · • • • · • · • ···· • · · « • · • · · · • · · · ·

with the proviso that when _Rg is alkoxycarbonyl of 1 to 5, C atoms in the alkoxy part or a benzyloxycarbonyl lskupinu, then R ₃ is a group

5-halo, 5-C 1 -C 4 -alkyl or 5-cyano and R ₄ is (phenyl) (hydroxy) C 1 -C 4 alkyl, (phenyl) (alkoxy) C 1 -C 4 alkyl C 1 -C 4 alkoxy, C 1 -C 4 alkyl, hydroxymethyl or C 1 -C 2 alkyl wherein Ar is thiene-2- or phenyl substituted

-3-yl, fur-2- or -3-yl or wherein said Ar moiety is optionally one or two substituents independently selected from the group consisting of halogen, provided that when R ₄ is benzyl and R ₅ is methyl, then R ₁₂ is not 4-hydroxy-piperidin-1-yl or when _R4 is benzyl and _R5 is methyl, then R is not the group of formula _g, C (O) N (CH _{3) 2,} with the proviso that when R? and R ₁₀ and R ₃₁ are H, then R ₄ is not imidazol-4-ylmethyl, 2-phenylethyl or 2-hydroxy-2-phenylethyl, with the proviso that when both of R ₈ and _Rg are n-pentyl, then represents 5-chloro, 5-bromo, 5-cyano,

(C 1 -C 5) -alkyl, (C 1 -C 5) -alkoxy or trifluoromethyl, provided that when R ₁₂ is 3,4-dihydroisoquinol-2-yl, the 3,4-dihydroisoquinol-2-one; -yl is not substituted by carboxyalkyl having 1 to 4 carbon atoms in the alkyl moiety,

• · · · · · e · · • • • • • • • • • · · · • • • · • • • • • · • · • ···· • · • • · · · · · · • • ·

with the proviso that when R is hydrogen and R _g is C 1 -C 6 alkyl, then R _g is a hydrogen atom, which is attached to the nitrogen atom of NHR _g, substituted by carboxy or alkoxycarbonyl having 1 to 4 carbon atoms alkoxy, with the proviso that when _Rg is COOH and each of R, R _10, rji and R 5 is H, then _R4 is not benzyl, H, (phenyl) (hydroxy) methyl, methyl, ethyl or an n-propyl group, and pharmaceutically acceptable salts and prodrugs thereof.

A first group of preferred compounds are those compounds of formula IA wherein

R1 is 5-H, 5-halo, 5-methyl,

5-cyano or 5-trifluoromethyl; ^R 10 ^{and R} 11 P ^re ďstavuje each independently hydrogen or halogen,

A is -C (H) =,

R ₂ and R ₃ are H;

R ₄ is hydrogen, methyl, phenylalkyl of 1 to 2 carbon atoms in the alkyl moiety, wherein said phenyl is substituted with one or two substituents independently selected from the group consisting of hydrogen and halogen, alkyl of 1 to 4 carbon atoms, (C až-C) alkoxy groups β · · β β β β β β β β β β β β β β β β β β 1 Trifluoromethyl, hydroxy, amino and cyano, and wherein R ₄ is optionally additionally substituted with one halogen atom, or

R ₄ is thien-2- or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, pyrid-2-, -3 or -4-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, thiazol-2-, -4- or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, imidazol-2-, -4- or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, fur-2- or -3-ylalkyl of 1 moiety up to 2 carbon atoms in the alkyl moiety, pyrrol-2- or -3-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, oxazol-2-, -4- or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, pyrazol-3-, -4 or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, isoxazol-3-, -4- or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, isothiazol-3-, - 4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, pyridazin-3- or -4-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, pyrimidine-2-, -4-, -5- or - 6-ylalkyl having 1 to 2 alkyl atoms, pyrazine-2- or -3-alkyl of 1 to 2 carbon atoms in the alkyl moiety or 1,3,5-triazin-2-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, the heterocyclic moiety present in the alkyl moiety; R ₄ is optionally substituted on carbon by one or two substituents independently selected from the group consisting of halogen, trifluoromethyl, C 1 -C 4 alkyl,

• B · · · · • · · • • • • • • • · · • · · · · • • • · * t • · • · · • • ···· • · · • • • · • B • • · • · • · • · • ·· ·

(C 1 -C 4) alkoxy, amino, and hydroxy, R ₅ is hydrogen,

R 8 is C (O) NR 8 R 8 or C (O) R ₁₁

Of the above-mentioned first group of preferred compounds, more preferred compounds are

first a group of particularly preferred compounds of the general formula

of formula IA, wherein

^R 4 is hydrogen, phenylalkyl having 1 to 2 carbon atoms in the alkyl moiety, thien-2 or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, fur-2- or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety wherein each ring in R ₄ is substituted with one or two substituents independently selected from the group consisting of hydrogen and fluorine atoms, ^R 6 represents C (O) R ₁₂ , ^R 12 represents morpholino, thiomorpholino, 1-oxothiomorpholino, 1,1-dioxothiomorpholino- pin, thiazolidin-3-yl, 1-oxothiazolidin-3-yl, 1,1-dioxothiazolidin-3-yl, pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl, piperazin-4-yl, azetidin-1-yl, 1,2-oxazinan-2-yl, isoxazolidin-2-yl, isithiazolidin-2-yl, 1,2-oxazetidin-2-yl, oxazolidin-3-yl, 1,3-dihydroisoindol-2-yl or azepan-1-yl-

• · · · • e • • · • • · • • · • • · · · • · · · · • · • · • · • • · • · • · · ···· • · · • • · • · • · • • · • • · · · · · • · · · · ·

wherein each R ₁₂ ring is optionally substituted with one or two substituents independently selected from the group consisting of halogen, C 1 -C 5 alkyl, C 1 -C 5 alkoxy, hydroxy, amino, mono- N- and di-N, C 1 -C 5 -alkylamino groups in each of the alkyl portions, formyl, carboxy, carbamoyl, mono-N- and di-N, N-C 1 -C 5 alkylcarbamoyl in each of the alkyl portions, (C 1 -C 5) alkoxycarbonyl, (C 1 -C 5) hydroxyalkyl, (C 1 -C 4) aminoalkyl, (C 1 -C 4) mono-N- and di-N, N-alkylamino (C 1 -C 4) alkylamino group , oxo, hydroxyimino and (C 1 -C 6) alkoxyimino, with the proviso that only heterocycles in R _{12 are} selected from the group consisting of thiazolidin-3-yl, pyrrolidin-1-yl couplings, piperidin-1-yl, piperazin-1-yl, piperazin-4-yl, azetidin-1-yl, 1,2-oxazinan-2-yl, isoxazolidin-2-yl and oxazolidin-3-yl are optionally substituted one or two substituents independently selected from the group consisting of oxo, hydroxyimino and (C 1 -C 6) alkoxyimino, and wherein the rings in R ₁₂ are optionally additionally substituted with one or two substituents independently selected from the group consisting of C 1 -C 6 alkyl 5 carbon atoms.

Of the above-mentioned first group, particularly preferred

• · • A • · • · · • • • • • • • • • · · • • · · · • • • · • • • • • • · • · • ···· • · • • • · • · • • • • · • • · · · · · • · · · · ·

The compounds are preferred to particular compounds selected from the group consisting of:

5-Chloro-1H-indole-2-carboxylic acid (1S) -benzyl-2- (3-hydroxyiminopyrrolidin-1-yl) -2-oxoethyl-7-amide, η 2- (cis-3,4-dihydroxypyrrolidin-1) 5-chloro-1H-indole-2-carboxylic acid 2-oxoethyl-7-amide, 5-chloro-1H-2 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) -2-oxoethyl] amide 5-Chloro-1H-indole-2-carboxylic acid, indole-2-carboxylic acid, N - (1S) -benzyl-2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2-oxoethyl 17-amide; 5-chloro-1H-indole-2-carboxylic acid- (1,1-dioxothiazolidin-3-yl) -2-oxoethyl] amide 5-chloro-1H-indole (2-oxo-2-thiazolidin-3-ylethyl) amide 5-Chloro-1H-indole-2-carboxylic acid, 2- (4-fluoro-benzyl) -2- (4-hydroxy-piperidin-1-yl) -2-oxo-ethyl] -amide,

5-chloro-1H-indole-2-carboxylic acid kyseliny (1S) -benzyl-2 - ((3RS) -hydroxypiperidin-1-yl) -2-oxoethyl} amide, ε 2-oxo-2 - ((1RS)) 7-Chloro-1H-indole-2-carboxylic acid-oxo-1-thiazolidin-3-yl) -ethyl amide,

5-Chloro-1H-indole-2-carboxylic acid C (1S) - (2-fluorobenzyl) -2- (4-hydroxypiperidin-1-yl) -2-oxoethyl] amide, E (1S) -benzyl-2- ( 5-Chloro-1H-indole-2-carboxylic acid (3S, 4S) -dihydroxypyrrolidin-1-yl) -2-oxoethyl-amide,

• · · · · · • · · • • • • • • • • • · · • • · · · • • • · • • • • · • · · • • ···· • · • • • · • · • · • • • • • · • · · · • · · · · ·

5-Chloro-1H-indole-2-carboxylic acid 1S) -benzyl-2- (3-hydroxyazetidin-1-yl) -2-oxoethyl] amide; (1S) -Benzyl-2- (3-hydroxyiminoazetidine) 5-chloro-1H-indole-2-carboxylic acid-1-yl) -2-oxoethyl iamide; and

5-Chloro-1H-indole-2-carboxylic acid (1S) -benzyl-2- (4-hydroxy-imino-piperidin-1-yl) -2-oxo-ethyl-amide.

Among the aforementioned group of particularly preferred compounds, the first group of the most preferred compounds are preferred, wherein r ₄ is hydrogen and r ₁₂ is thiazolidin-3-yl, 1-oxothia zolidin-3-yl, 1,1-dioxothiazolidin-3- or R ₁₂ are optionally substituted by one or two radicals independently selected from the group consisting of carboxy, C 1 -C 5 alkoxycarbonyl, C 1 -C 3 hydroxyalkyl, aminoalkyl (C 1 -C 3) mono-N- and di-N, N-alkylamino (C 1 -C 3) alkyl group in each alkyl moiety; or

R ₁₂ is pyrrolidin-1-yl substituted with one or two substituents independently selected from the group consisting of carboxy, C 1 -C 5 alkoxycarbonyl, C 1 -C 5 alkoxy, hydroxy, C 1 -C 3 hydroxyalkyl carbon atoms, amino groups, aminoalkyl groups

· · • · · · • • · • • • • • • • • · · • • · · · • • • · • • • • • · • · • ···· • · • • · · • · · · • • · · ·

C 1 to 3 carbon atoms, C 1 -C 3 mono-N- and di-N, N-alkylaminoalkyl groups in each of the alkyl moieties, and C 1 -C 4 -alkoxy N- and di-N, N-alkylamino groups in each and the rings in R ₁₂ are optionally additionally substituted with two substituents independently selected from the group consisting of C 1 -C 5 alkyl.

Of the immediately preceding group of the most preferred compounds, one can transfer it to the compounds where

a. R ₃ is 5-chloro, R ₁₀ and R _{1; L} are hydrogen and R ₁₂ is cis-3,4-dihydroxypyrrolidin-1-yl,

b. R 11 is 5-chloro, R ₁₀ and R ₃₁ are hydrogen and R ₁₂ is (3S, 4S) -dihydroxypyrrolidin-1-yl,

c. R j is 5-chloro, R _10, and R _{i, L} is H and R ₁₂ is 1, l-dioxo-thiazolidin-3-yl,

d. R is 5-chloro, R _10, and R _{i, L} is H and R ₁₂ is thiazolidin-3-yl, and

e. R 1 is 5-chloro, R ₁₀ and R 6 are hydrogen and R ₁₂ is 1-oxothiazolidin-3-yl.

Of the aforementioned group of particularly preferred compounds, the compounds of the second group of the most preferred compounds are preferred, wherein: · represents phenylmethyl, thien-2-3-ylmethyl, wherein the rings in R ₄ are substituted with one or two atoms and

r ₄ ^r 12 ^R 12 12 · ^R _{4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4} Or optionally fluorine, is thiazolidin-3-yl, 1-oxothiazolidin-3-yl, 1,1-dioxothiazolidin-3-yl or oxazolidin-3-yl, wherein the R ₁₂ substituents are optionally substituted by one or carboxy carbon up to 3 carbon atoms, aminoalkyl carbon, mono-N- and di-N, N-alkylaminoalkyl of 1 to 3 carbon atoms in each of the alkyl moieties, or two radicals independently selected from or alkoxycarbonyl of 1 to 5 atoms in the alkoxy moiety, hydroxyalkyl with 1 to 3 atoms is azetidin-1-yl, pyrrolidin-1-yl, or piperidin-1-yl, each of which is substituted with one or two substituents independently selected from the group consisting of carboxy, alkoxycarbonyl up to (C 1 -C 5) hydroxyalkyl of aminoalkyl mono-N 1 to 3 alkoxy carbon atoms with 1 and di-N, N-alkylaminoalkyl carbon atoms in each of the hydroxy, alkoxy, amino, mono-N- and up to 5 carbon atoms of the moieties, moiety, carbon, carbon, pins with alkyl moieties, s (C 1 -C 5) di-N, N-alkylamino group (s) in each of the alkyl moieties, oxo, hydroxyimino and (C 1 -C 5) alkoxyimino groups, and R ₁₂ rings are optionally substituted with additionally one or two substituents independently selected from the group consisting of from alkyl groups

· · · · · · • · · • • • • • · • • · · · • • · · · • · • · • · • • • • · · • · ···· • · · • • • · • · · · • · · • · ·

C 1 -C 5.

Of the immediately preceding group of the most preferred compounds, compounds are preferred

a. R is 5-R ₁₀ and R _{i, l} are H; _R4 is 4-fluorobenzyl, R ₁₂ is

4-hydroxypiperidin-1-yl and stereochemistry of carbon atom (a) is (S),

b. R 1 is 5-chloro, R ₁₀ and R _{1; L} are hydrogen, R ₄ is benzyl, R ₁₂ is 3-hydroxy-piperidin-1-yl, and the stereochemistry of the carbon atom (a) is (S),

c. R ₃ is 5-chloro, R ₁₀ and R are hydrogen, R ₄ is benzyl, R ₁₂ is cis-3,4-dihydroxypyrrolidin-1-yl, and the carbon atom stereochemistry of (a) is (S),

d. R 1 is 5-chloro, R ₁₀ ^{and R 11} are hydrogen, R ₄ is benzyl, R ₁₂ is 3-hydroxyiminopyrrolidin-1-yl, and the stereochemistry of the carbon atom (a) is (S),

e. R ₁ is 5-chloro, R _10, and R _{i, L} is H, R ₄ is 2-fluorobenzyl, R ₁₂ predstaavuje

4-hydroxypiperidin-1-yl and the stereochemistry of carbon atom (a) is (S),

f. R is 5-chloro, R _10, and R _{i, l} are H; _R4 is benzyl, _R12 is (3S, 4S) -dihydroxy-l-yl; and the stereochemistry of carbon (a) is (S).

· · · · · · • · · • • • • • • • • • · · • • · · · • • • · • • • • · • · • · • ···· • · • • • • • · • • • • • • · · · · · · • · · · · ·

g. is 5-chloro, R @ ₁₀ and R @ _{1, L} are H; _R4 is benzyl, _R12 is 3-hydroxyazetidin-l-yl; and the stereochemistry of carbon (a) is (S).

h. R 5 is 5-chloro, R ₁₀ and R ₁₀ are hydrogen, R ₄ is benzyl, R ₁₂ is 3-hydroxyiminoazetidin-1-yl, and the carbon atom stereochemistry of (a) is (S), and

i. R 5 is 5-chloro, R ₁₀ and R 22 are hydrogen, R ₄ is benzyl, R ₁₂ is 4-hydroxyiminopiperidin-1-yl, and the carbon atom stereochemistry of (a) is (S).

Of the aforementioned first group of preferred compounds, more preferred compounds are those belonging to the second group of particularly preferred compounds of formula IA wherein r ₄ is hydrogen, phenylalkyl of 1 to 2 carbon atoms in the alkyl moiety, thien-2- or -3-ylalkylsupine C 1-2 alkyl, fur-2- or -3-ylalkyl C 1-2 alkyl, wherein the rings in R ₄ are substituted with one or two substituents independently selected from the group consisting of atoms hydrogen and fluorine,

R is a group of formula C (O) NR _g R _9, and

R ₈ is hydrogen, C 1 -C 5 alkyl, hydroxy or C 1 -C 4 alkoxy, and

· · · · · · • · · • • • • • • • · · • • · · · • • • · • · • • • · • · • ···· • · · • · • · • · • • · · · · · · · • ·· ·

R _g is H, cyklalkylskupinu having 4 to 6 carbon atoms, cycloalkylalkyl having 3 to 6 carbon atoms in the cycloalkyl and C 1 -C 5 alkyl, methylene-perfluorinated C 1 -C 3 alkyl, pyridyl, pyrrolidinyl , oxazolilyl, thiazolyl, imidazolyl, peridyl, benzothiazolyl or thiochromanyl, or

R ₉ is C 1 -C 5 alkyl optionally substituted with a substituent selected from the group consisting of cycloalkenyl of 4 to 6 carbon atoms, phenyl, thienyl, pyridyl, pyrrolidinyl, oxazolyl, thiazolyl, imrazazolyl, morpholinyl, thiomorpholinyl, 1-oxothiomorpholinyl and 1,1-dioxothiomorpholinyl, said C 1 -C 5 alkyl or C 1 -C 4 alkoxy optionally further substituted by one or two substituents independently selected from the group consisting of halogen, hydroxy, C 1 -C 5 alkoxy, amino, mono-N- and di-N, N-C 1 -C 5 alkylamino in each of the alkyl moieties, cyano, carboxy, and C 1 -C 4 alkoxycarbonyl in the alkoxy moiety, and wherein each of the rings in Rg is optionally substituted on carbon by one or two substituents independently selected from the group consisting of halogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, hydroxy, amino, mono-N- and di-N, N- (C 1 -C 4) alkylamino, carbamoyl, (C 1 -C 5) alkoxycarbonyl, and carbamoyl.

· · · · · · • ·· · • · • • · • • · · · • · · · · • · • · • · • • · • · · • · ···· • · · • • · • · • · • • · • · · • e · · • · · · · ·

Of the immediately preceding second group of particularly preferred compounds, compounds wherein

a. R 1 is 5-chloro, R ₁₀ is hydrogen, R ₄ is benzyl, R _g is methyl, R _g is 3- (dimethylamino) propyl,

b.stereochemistry at carbon (a) is (S),

Rj represents

R ₄ represents

R ₉ represents

5-chloro, and R Rjq · ^ are hydrogen, benzyl, R _g is methyl, and

3-pyridyl,

c. the stereochemistry of the carbon atom (a) is (S),

R is 5-chloro, _R10 and Rn are H; _R4 is benzyl, R _g is methyl and R is 2-hydroxyethyl, and

d. the stereochemistry of the carbon atom (a) is (S),

R is 5-fluoro, ^R io ^{and Rn} are H; _R4 is 4-fluorophenylmethyl, R _g is methyl, and R _g is 2-morfolínoetylskupinu.

A third group of particularly preferred compounds are those falling within the first group of preferred compounds wherein:

R ₄ is hydrogen, phenylalkyl of 1

· · · · · · • · · « • • • • • • • • · · • • · · · * • • · • • • • • • · · • • ···· • · • • • • • · • • • • • • · · • · · · • • · · · ·

up to 2 carbon atoms in the alkyl moiety, thien-2- or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, fur-2- or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, the rings in R being ₄ are substituted with one or two substituents independently selected from the group consisting of hydrogen and fluorine atoms,

R ₆ is a group of formula C (O) NR _g R g, and

R 8 is hydrogen, C 1 -C 5 alkyl, hydroxy or C 1 -C 4 alkoxy, and

R _g is C 1 -C 4 alkoxy optionally substituted with a substituent selected from the group consisting of C 4 -C 6 cycloalkenyl, phenyl, thienyl, pyridyl, pyrrolidinyl, oxazolyl, thiazolyl, imidazolyl, imidazolyl, imidazolyl morpholinyl, thiomorpholinyl, 1-oxothiomorpholinyl and 1,1-dioxothiomorpholinyl, wherein said C 1 -C 5 alkyl or C 1 -C 4 alkoxy is optionally additionally substituted with one or two substituents independently selected from the group consisting of hydroxy, halogen C 1 -C 5 alkoxy, amino, mono-N- and di-NN-C 1 -C 5 -alkylamino in each of the alkyl moieties, cyano, carboxy and C 1 -C 4 alkoxycarbonyl, and ···· · ·· • ···· · · · · · · · • ···· * · · · · · · · • • · · · · · • · · · · ····

9 · · · · · · · · each of the rings in R _g is alkyl optionally substituted by one or two substituents independently selected from the group consisting of halogen, C 1 -C 4 alkyl, C 1 -C 4 alkyl hydroxy, amino, mono-N- and di-N, (C1-C4) alkylamino, carbamoyl, (C1-C5) alkoxycarbonyl and carbamoyl.

Of the immediately preceding third group of particularly preferred compounds, compounds are preferred

a. R 1 is 5-chloro, R ₁₀ and R 6 are hydrogen, R ₄ is benzyl, R _g is methyl, R _g is 2-hydroxyethoxy,

b. the stereochemistry of the carbon atom (a) is (S),

R is 5-chloro, R _10, and R ^ are H; _R4 is 4-fluorophenylmethyl, R _g, R g is methyl, and methoxy pre-melted, and

c. the stereochemistry of carbon (a) is (S), 5-chloro Rjpredstavuje, R _10, and R _{1, L} are H; _R4 is benzyl, R _g is methyl and R is methoxy.

A second group of preferred compounds of formula IA are those wherein

R! represents a 5-halogen, 5-methyl, 5-cyano, or trifluoromethyl group; and represents a 5-halogen, 5-methyl, 5-cyano, or trifluoromethyl group; · a aaaa aa aaaa ·· ·· aa aa aa aa and ^R 10 and R _1X are each independently hydrogen or halogen, is -C (H) =, ^R 2 ^are 3 are H, is H, C 1 -C 2 -alkyl phenylalkyl, C 1 -C 2 -alkyl or thien-2- or -3-ylalkyl, C 1 -C 2 -alkyl or C 2 -C 2 -alkyl, where each of the above rings is independently substituted with one or two hydrogen or fluorine atoms,

R represents a hydrogen atom and represents an alkoxycarbonyl group having 1 to 5 carbon atoms in the alkoxy moiety.

A third group of preferred compounds of formula IA are those wherein

^R 1 represents 5-halogen, cyano or trifluoromethyl, 5-methyl, 5- ^R 10 ^{and R} 11 each independently represents a halogen atom, hydrogen or A represents -C (H) =, ^{R2 and R3} represent hydrogen atoms, ^R 4 is hydrogen, methyl or

·····························

phenyl of 1 to 2 carbon atoms in the alkyl moiety, wherein said phenyl is substituted with one or two substituents independently selected from the group consisting of hydrogen, halogen, (C 1 -C 4) alkyl, (C 1 -C 4) alkoxy, tri fluoromethyl, hydroxy, amino and cyano, and wherein said phenyl is optionally additionally independently substituted with one or two hydrogen or halogen atoms, or is thiene-2- or -3-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, pyrid-2 -, -3- or -4-yalkyl having 1 to 2 carbon atoms in the alkyl moiety, thiazol-2-, -4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, imidazole-2-, -4 or -5-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, fur-2- or -3-ylalkyl of 1 to 2 carbon atoms in the alkyl moiety, pyrrol-2- or -3-ylalkyl of 1 to 2 atoms an alkyl moiety, an oxazol-2-, -4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, a pyrazol-3-, -4- or -5-ylalkyl having 1 to carbon atoms in the alkyl moiety, isoxazol

3-, -4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, isothiazol-3-4- or -5-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety pyridazin-3- or -4-ylalkyl C 1 -C 2 alkyl, pyrimidin-2-, -4- or -5-alkyl C 1 -C 2 alkyl, pyrazin-2- or -3-ylal ·· ·· ·· · ·· · 1 to 2 carbon atoms in the alkyl moiety or 1,3,5- triazin-2-ylalkyl having 1 to 2 carbon atoms in the alkyl moiety, wherein the heterocyclic radical in R ₄ is optionally substituted on carbon by one or two substituents independently selected from the group consisting of halogen, trifluoromethyl, alkyl of 1 to 4 carbon atoms , (C 1 -C 4) alkoxy, amino and hydroxy groups,

R ₅ represents a hydrogen atom and

R 8 is carboxy.

Of this third group of preferred compounds, preference is given to compounds belonging to the first group of particularly preferred compounds wherein ^R 10 ^and

R1 _and R2 are hydrogen atoms and

R ₄ represents a hydrogen atom.

Among the preferred compounds, R 1 is 5-chloro.

immediately preceding i, a particular group of compounds is preferred, wherein

Another aspect of the mammal afflicted with the condition is that the insulin resistance administered by the invention is a resistance to a mammal afflicted with a therapeutically effective amount of a method of treating insulin,

a. a first compound, wherein the first compound is an aldose reductase inhibitor; and, ··· · ··························

b. a second compound, wherein the second compound is a glycogen phosphorylase inhibitor.

Examples of insulin resistance states, either individually or in a group, are preferably diabetes, hyperinsulinemia, impaired glucose tolerance, hyperglycemia and / or post-meal hyperlipidemia, type II diabetes, altered body composition, decreased muscle mass, which essentially containing fat, obesity (especially abdominal vicseral obesity), hypertension, dyslipidemia (e.g., elevated levels of free fatty acids, triglycerides, VLDL cholesterol and LDL chloesterol, and lowered HDL chloesterol), atherosclerosis, tissue ischemia, obesity, is also referred to as metabolic syndrome), pregnancy, infectious conditions, uremia, hyperadrogenism, hypercortisolemia and other conditions of excess adrenocortical hormones, acromegaly, excess growth hormone or polycystic ovaries.

Insulin resistance is particularly preferred for diabetes.

The aldose reductase inhibitor is preferably 3,4-dihydro-4-oxo-3- [5- (trifluoromethyl) -2-benzothiazolyl] methyl] -1-phthalazineacetic acid and a pharmaceutically acceptable salt thereof.

A preferred glycogen phosphorylase inhibitor is 5-chloro-1H-indole-2-carboxylic acid [(1S) -benzyl- (2R) -hydroxy-3 - ((3S) -hydroxypyrrolidin-1-yl) -3-oxopropyl] -amide; 5-Chloro-1H-indole-2-carboxylic acid (2S) -benzyl-3 - ((3S, 4S) -dihydropyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide, ·· ·· ··· · ····················································· (1S) - ((R) -Hydroxymethylcarbamoylmethyl) -2-phenylethyl] -amide

5-chloro-1H-indole-2-carboxylic acid 5-chloro-1H-indole-2-carboxylic acid [(1S) - (R) -hydroxymethoxymethylcarbamoyl) methyl) -2-phenyl-ethylamide,

5-Chloro-1H-indole-2-carboxylic acid L (1S) - ((R) -hydroxy- (2-hydroxyethyl) methylcarbamoylmethyl) -2-phenylethyl] amide, E (1S) -benzyl-2- (3) 5-Chloro-1H-indole-2-carboxylic acid-hydroxy-pyrrolidin-1-yl) -2-oxo-ethyl] -amide, [2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2-oxo-ethyl] -amide

5-Chloro-1H-5-chloro-1H-indole-2-carboxylic acid, (1S) -benzyl-3 - ((cis) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide 5-chloro-1H-indole-2-carboxylic acid [2 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) -2-oxoethyl] indole-2-carboxylic acid, indole-2-carboxylic acid, [(1S) -benzyl-2] - 5-chloro-1H-indole-2-carboxylic acid (cis-3,4-dihydroxypyrrolidin-1-yl) -2-oxoethyl amide,

5-Chloro-1H-indole-2-carboxylic acid E2- (1,1-dioxothiazolidin-3-yl) -2-oxoethyl] amide, N (1S) - (4-fluorobenzyl) -2- (4-hydroxypiperidin-1-yl) yl) 5-chloro-1H-indole-2-carboxylic acid 2-oxoethyl] amide, 5-chloro-N (1S) -benzyl-2 - ((3RS) -hydroxypiperidin-1-yl) -2-oxoethyl] amide 5-Chloro-1H-indole-2-carboxylic acid -1H-indole-2-carboxylic acid, 2-oxo-2 - ((1RS) -oxothiazolidin-3-yl) ethyl] amide, or [(1S) -benzyl] 5-Chloro-1H-indole-2-carboxylic acid 2- (3-hydroxyazetidin-1-yl) -2-oxoethyl-7-amide.

In a particularly preferred embodiment, the mammal is a male or female human.

Further preferred is an embodiment wherein the first compound and the second compound are administered substantially simultaneously.

It is a further object of the present invention to provide a synergistic pharmaceutical composition for enhancing insulin sensitivity in a mammal comprising:

a. an amount of the first compound which is an aldose reductase inhibitor; and

b. the amount of the second compound which is a glycogen phosphorylase inhibitor wherein the amount of the first compound alone and the amount of the second compound alone is not sufficient to increase insulin sensitivity when administered alone with the combined effect of the amount of the first compound and the amount of the second compound is greater than the sum of the insulin sensitivity enhancing effects which may be achieved with the amount of the first and second compounds separately, and a pharmaceutically acceptable diluent or carrier.

A further object of the present invention is a kit, the nature of which is to comprise a kit

and a therapeutically effective amount of an aldose reductase inhibitor and a pharmaceutically acceptable carrier in a first unit dosage form, and a pharmaceutically acceptable carrier in a first unit dosage form; · · · ···· · · ·················

b. a therapeutically effective amount of a glycogen phosphorylase inhibitor and a pharmaceutically acceptable carrier in a second unit dosage form, and

c. a container for said first and second dosage forms.

It is a further object of the present invention to provide a synergistic method for increasing insulin sensitivity in a mammal suffering from an insulin resistance condition comprising administering to said mammal such a mammal.

a. an amount of the first compound which is an aldose reductase inhibitor; and

b. the amount of the second compound which is a glycogen phosphorylase inhibitor, wherein the amount of the first compound alone and the amount of the second compound alone is not sufficient to increase insulin sensitivity when administered alone, and the combined effect of the amount of the first compound and the amount of the second compound is greater than the sum insulin that can be achieved with the amounts of the first and second compounds separately.

A further object of hovering is to provide a method of reducing tissue damage that is or could be caused by ischemia, which comprises administering to a mammal in need of such treatment a therapeutically effective amount.

a. aldose reductase inhibitor a

b. a glycogen phosphorylase inhibitor.

Preferred ischemic tissues, taken individually or as a group, are ischemic tissues of the heart, brain, liver, kidney of the lung, intestine, skeletal muscle, spleen, pancreas, nerves, spinal cord, retina, vasculature or intestinal tissue.

A particularly preferred tissue is heart tissue.

The combination of the invention is prophylactic.

preferably administered

Ischemia damage treatable according to the invention may occur during organ transplantation.

Combination by heart surgery.

The invention is preferably administered prior to resistance

Conditions (syndrome or condition of tissue sensitivity and / or mormal resistance).

an insulin condition; insulin), the insulin status of the organism i leads to cell responsiveness (or

This resistance to glucose, protein metabolism is understood to be a condition in which there are numerous abnormalities in lipids, electrolyte and ion imbalances, and growth of, for example, organs, tissues and cells in organs, mammals compared to insulin sensitivity), which may manifest as one or more the number of conditions including, but not limited to, hyperinsulinemia, impaired glucose tolerance (IGT), hyperglycemia and / or hyperlipidemia after a meal, type II diabetes, altered body composition, loss of fat-free muscle tissue, obesity (especially abdominal visceral obesity), hypertension, dyslipidemia (e.g., elevated levels of free fatty acids, triglycerides, VLDL cholesterol and LDL cholesterol, and decreased HDL cholesterol), atherosclerosis, tissue ischemia, and cardiovascular disease (Kopelman and Albon, 1997, DeFronzo and Ferrannini, 1991, Reast, 1991, Reaven, 1991) et al., 1997).

· · · · · · • · · • • • • • · • • • • · • • · · · • · • · • • • • • • · · • · ···· • · • • • • • · • · • • · • · · · · · · • • · · · ·

As a result of the insulin resistance state, a greater amount of insulin is required to access or achieve the same biological effect of insulin in organs, tissues and cells compared to normal melting (or insulin sensitivity state). This leads to an increased demand for insulin secretion (aggravated hyperinsulinemia) in the pancreas, and in extreme cases, pancreatic failure and insulin deficiency leading to diabetic I. Insulin resistance states may include, for example, obesity, syndrome X (also referred to as metabolic syndrome) , pregnancy or infectious conditions, uremia, hyperdrogenism, hypercortisolemia and other conditions of excess adrenocortical hormones, acromegaly, excess growth hormone or polycystic ovaries, or may be associated with older age or specific ethnic groups (Kopelman and Albon, 1997).

Insulin sensitivity enhancing effect is understood to mean the effect that causes the patient's tissues to produce a normal or better than normal biological response to a given amount of insulin.

In addition to substantially complete prevention, the term " reduction " also includes partial prevention which is less than 100% but higher than the results obtained with placebo or drug administration.

Ischemia-induced tissue damage refers to a stay directly associated with a reduction in blood supply to the tissue, for example due to a clot or blood vessel obstruction that supplies the tissue with blood, i.e. it leads to a decrease in the oxygen supply of this tissue, a deterioration of its performance, dysfunction and necrosis. alternatively, when blood flow and organ perfusion are quantitatively adequate, it may

• · • · • · • • • • • • • • · • · • • · · · • • • · • · • • • · • · • ···· • · · • • · • · · · • · · • ·

the capacity of the blood or perfusion medium to carry oxygen may be reduced, for example in a hypoxic environment, so that oxygen supply to the tissues is reduced, and consequently performance failure, tissue dysfunction and tissue necrosis occur.

The term aldose reductase inhibitor refers to compounds that inhibit the bioconversion of glucose to sorbitol catalyzed by the enzyme aldose reductase.

By glycogen phosphorylase inhibitor is meant any substance, agent or combination of substances and / or agents reducing, retarding or eliminating the enzymatic action of glycogen phosphorylase. The enzymatic action of glycogen phosphorylase, which is currently known, is based on the degradation of glycogen by catalyzing the reversible reaction of the macromolecule of glycogen and inorganic phosphate to glucose-1-phosphate and the macromolecule of glycogen which is one glucosyl residue shorter than the original macromolecule in the direction of glycogenolysis).

The term treatment also includes preventive (i.e. prophylactic) and palliative treatments.

The adjuvant pharmaceutically suitable is used to indicate carriers, diluents, excipients and / or salts, which must be compatible with the other ingredients of the formulation and do not harm the recipient.

Prodrugs are prodrug compounds that, when administered by a chemical or physiological mechanism, release the drug in vivo (for example, the prodrug is exposed to physiological pH or converted to the desired form of the drug by enzymatic treatment). ···································································· ·

99

99999). Such prodrugs, for example, liberate the corresponding νοϊηύ acid upon cleavage.

The term alkylene refers to a straight or branched chain saturated hydrocarbon radical from which both terminal carbon atoms are removed from the hydrogen atom. Examples of such groups (which take into account a given chain length) include methylene, ethylene, propylene, butylene, pentylene, hexylene, and heptylene.

The term halogen refers to chlorine, bromine, iodine or fluorine.

The term alkyl refers to a straight or branched chain saturated hydrocarbon radical. Examples of such alkyl groups (which take into account the specified chain length) include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neoprentyl, tert-pentyl-, 1-methylbutyl-, 2-methylbutyl-,

3-methylbutyl-, hexyl-, isohexyl-, heptyl- and octyl.

The term alkoxy refers to a straight or branched chain saturated alkyl group which is bonded via an oxygen atom. Examples of such alkoxy groups (which take into account the specified chain length) include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, isopentoxy, neopentoxy, tert. pentoxy-, hexoxy-, isohexoxy-, heptoxy- and octoxy.

In the aforementioned mono-N- or di-N, N-alkyl groups having 1 to x carbon atoms in each of the alkyl portions (where x represents a number), the number of carbon atoms of each of the alkyl portions is taken independently.

If the carbocyclic or heterocyclic radical may be bonded or otherwise attached to said substrate via different ring atoms and a particular point of such attachment is not mentioned, then all possible attachment points, whether by a carbon atom or a trivalent nitrogen atom, are possible. Thus, for example, pyridyl means 2-, 3- or 4-pyridyl, thienyl-2-buto-3-thienyl, and the like.

Pharmaceutically acceptable salts are those containing non-toxic anions. Non-limiting examples of such salts include chloride, bromide, iodide, sulfate, hydrogensulfate, phosphate, acetate, maleate, furmate, oxalate, lactate, tartrate, citrate, gluconate, methanesulfate, and the like.

4-toluenesulfonate. Pharmaceutically acceptable salts also include salts with non-toxic cations. Such salts include, but are not limited to, those containing sodium, potassium, calcium, magnesium, ammonium, or protonated benzatin (N, N'-dibenzylethylenediamine), choline, ethanolamine, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) cations (N-benzylphenylethylamine), piperazine or tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol).

As used herein, a reaction-inert solvent or inert solvent is a solvent or solvent mixture that does not interact with the starting materials, reagents, intermediates or products in a manner that yields the desired product which would be unfavorable to the product.

effects on

The plus signs in parentheses indicate the direction in which a particular stereoisomer rotates the plane of polarized light.

· · · · · · • · · • • • • • · • • • • · • • · · · • · • · • • • • • • · • · · ···· • · • • • · • · • · • • · • · · · · · · • · · · · ·

It will be clear to those skilled in the art of chemistry that certain compounds of the invention will contain one or more atoms that may be in particular stereochemical or geometric configurations to form stereoisomers and configuration isomers. All such isomers and mixtures thereof are the subject of this invention.

Also included within the scope of the invention are the hydrates and olates of the compounds of the invention.

DTT stands for dithiothreitol, DMSO stands for dimethylsulfoxide and EDTA stands for ethylenediaminetetraacetic acid.

Other features and advantages of the invention will be explained from the following detailed description and claims.

The compounds of the invention can be prepared using known methods, in particular as described herein. Some methods of making the compounds of the invention are further features of the invention and are illustrated in the following reaction schemes. Other methods may be described in the Examples.

Any aldose reductase inhibitor can be used as the compounds of the present invention. The term aldose reductase inhibitor refers to compounds which inhibit the bioconversion of glucose to sorbitol catalyzed by the enzyme aldose reductase. Such inhibition is readily determined by those skilled in the art using standard assays (J. Malone, Diabetes, 29: 861-864, 1980, Red Cell Sorbitol, an Indicator of Diabetic Control). Various aldose reductase inhibitors are listed below with appropriate references. However, other such substances will be known to those skilled in the art. US

· · · · • · · • • • • · • • · · • • · · · • · • · • · • • • · • · · ···· • · · • • • 9 • · • • · · · • · • · • ·· ·

patents are incorporated herein by reference to the relevant patent literature relating to the compounds, and are described using the USAN chemical nomenclature and other designations in parentheses, if applicable.

Tissue aldose reductase inhibitor activity can be determined by assaying the amount of aldose reductase inhibitor required to reduce tissue sorbitol (i.e., inhibit additional sorbitol production due to aldose reductase blocking) or reduce tissue fructose (inhibiting sorbitol production due to aldose reductase blocking) Although not wishing to be bound by any particular theory or mechanism, we believe that an aldose reductase inhibitor inhibits aldose reductase inhibits or reduces ischemia-related damage as described below.

Thus, examples of aldose reductase inhibitors useful in the compositions and methods of the invention include:

1. 3- (4-bromo-2-fluorobenzyl) -3,4-dihydro-4-oxo-1-phthalazineacetic acid (polnalrestat, US 4,251,528);

2. N - [(5-Trifluoromethyl) -6-methoxy-1-naphthalenyl] -1-thioxomethyl} -N-methylglycine (tolrestat, US 4 600 724);

3. 5- £ (Z, E) -6-Methylcinamylidene-4-oxo-2-thioxo-3-

thiazolideneacetic acid (epalrestate, U.S. Pat. No. 4,464,382, U.S. Pat

791 126, US 4,831,045),

4. 3- (4-bromo-2-fluorobenzyl) -7-chloro-3,4-dihydro-2,4-dioxo-1 (2H) -quinazoline acetic acid (zenarestat, US 4,734,419 and 4,883,800); ························

• · ····

5. 2R, 4R-6,7-dichloro-4-hydroxy-2-methylchroman-4-acetic acid (US 4,883,410),

6. 2R, 4R-6,7-dichloro-6-fluoro-4-hydroxy-2-methylchroman-4-acetic acid (US 4,883,410),

7. 3,4-dihydro-2,8-diisopropyl-3-oxo-2H-1,4-benzoxazol-4-acetic acid (US 4 771 050);

8. 3,4-Dihydro-3-oxo-4- (4,5,7-trifluoro-2-benzothiazolyl) methyl] -2H-1,4-benzothiazin-2-acetic acid (SPR-210, US 5,252,572),

9. N- (3,5-dimethyl-4- (nitromethyl) sulfonylphenyl) -2-methylbenzeneacetamide (ZD5522, US 5 270 342 and US 5 430 060);

10. (S) -6-fluorospiro £ chroπlan ^4,4, -imidazolidín7-2,5dión (sorbinil, U.S. 4,130,714).

11. d-2-methyl-6-fluorospiro (chroman-4 ', 4'-imidazolidine) -2', 5'-dione (US 4,540,704),

12. 2-fluorospiro (9H-fluorene-9,4'-imidazolidine) -

2 ’, 5’, -dion (US 4,438,272),

13. 2,7-difluorospiro (9H-fluorene-9,4'-imidazolidine) -2 ', 5'-dione (US 4,436,745, US 4,438,272)

14. 2,7-Difluoro-5-methoxyspiro (9H-fluorene-9,4'-imidazolidine) 2 ', 5'-dione (US 4,436,745, US 4,438,272)

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· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

15. 7-fluorospiro (5H-indenol-1,2-b pyridine-5,3'-pyrrolidine-2,5-dione (US 4,436,745, US 4,438,272);

16. ^{d-cis-6-chloro-1} 2 ', 3'-dihydro-2'-methyl spiro (imidazolidine-4, 4' * -4-H-pyrano (2,3-b) pyridine) -2,5 -dion (US 4 980 357),

17 £ spiro imidazolidine-4,5 * (6H) chinolínJ-2,5-dione-3'-chloro-7 ', ^8? -dihydro-7 ^; -methyl- (5'-cis) (US 5,066,659),

18. (2S, 4S) -6-fluoro-2 ', 5'-dioxospiro (chroman-4, 4'-imidazolidine) -2-carboxamide (US 5,447,946); and

19. 2- (4-Bromo-2-fluorophenyl) methyl] -6-fluorospiro-isoquinoline-4- (1H), 3'-pyrrolidine-1,2 *, 3,5 '(2H) tetrone ( ARI-509, US 5,037,831).

Examples of other aldose reductase inhibitors include compounds of Formula I

where

Z represents oxygen or sulfur,

R ¹ represents a hydroxy group or a group which can be removed in vivo to give a compound of the general formula I, wherein R ¹ represents a hydroxyl of a pin and

X and Y are the same or different and are each independently hydrogen, trifluoromethyl, fluorine or chlorine, and pharmaceutically acceptable salts thereof.

Of the above aldose reductase inhibitors, compounds of formula 1, 2, 3, 4, 5, 6, 9, 10 and 17 and the following compounds of formula I are preferred:

20. 3,4-Dihydro-3- (5-fluorobenzothiazol-2-ylmethyl) -4-oxophthalazin-1-ylacetic acidR ¹ = hydroxy, X = F, Y = HJ

21. 3- (5,7-Difluorobenzothiazol-2-ylmethyl) -3,4-dihydro-4-oxophthalazine-1-acetic acid [R ¹ = hydroxy, X = Y = FJ,

22. 3- (5-Chloro-benzothiazlo-2-ylmethyl) -3,4-dihydro-4-oxo-phthalazin-1-ylacetic acid R ¹ = hydroxy, X = Cl, Y = H 3,

23. 3- (5,7-Dichlorobenzothiazol-2-ylmethyl) -3,4-dihydro-

-4-oxophthalazin-1-ylacetic acid [r ¹ = hydroxy, X = Y =

C1J.

· · • · • · • · · • • • • • • • · • · • • · · · • • • · • · • • • · • · • ···· • · · • · · • · · · • • · • ·

24. 3,4-dihydro-4-oxo-3- (5-trifluoromethylbenzoxazol-2-ylmethyl) phthalazin-1-ylacetic acid R ¹ = hydroxy, X = CF ₃ , Y = H 3,

25. 3,4-dihydro-3- (5-fluorobenzoxazol-2-ylmethyl) -4-oxophthalazin-1-ylacetic acid R ¹ = hydroxy, X = F, Y = HJ;

26. 3- (5,7-Difluorobenzoxazol-2-ylmethyl) -3,4-dihydro-4-oxophthalazin-1-ylacetic acid R ¹ = hydroxy, X = Y = FJ,

27. 3- (5-chlorobenzoxazol-2-ylmethyl) -3,4-dihydro-4-oxophthalazin-1-ylacetic acid ^ R ¹ = hydroxy, X = Cl, Y = H 3,

28. 3- (5,7-dichlorobenzoxazol-2-ylmethyl) -3,4-dihydro-4-oxophthalazin-l-yl-acetic acid / R ¹ = hydroxy; X = Y = Clja

29. Zopolrestat, 3,4-dihydro-4-oxo-3- [5- (trifluoromethyl) -2-benzothiazolyl] methyl] -1-phthalazineacetic acid

R ¹ = hydroxy; X = trifluoromethyl; Y = H.

In compounds 20 to 23 and 29 Z is sulfur and in compounds 24 to 28 it is oxygen.

Of the above subgroup of preferred compounds, compounds 20 to 29 are more preferred, and compound 29 is particularly preferred.

The compounds that are aldose reductase inhibitors of the invention are readily available or can be readily synthesized by one skilled in the art using known synthetic procedures.

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organic compounds, especially with respect to the descriptions found in the relevant patent literature.

Any glycogen phosphorylase inhibitor can be used as the second compound of the invention. glycogen phosphorylase is understood or a combination of substances and / or

An inhibitor is any substance, agent of agents reducing, retarding or eliminating the enzymatic action of glycogen phosphorylase. The enzymatic action of glycogen phosphorylase, which is currently well known, consists in the degradation of glycogen by catalyzing the reversible reaction of the macromolecule of glycogen and inorganic phosphate to glucose-1-phosphate and the macromolecule of glycogen which is one glucosyl residue shorter than the original macromolecule in the direction of glycogenolysis). Such effects are readily determined by one skilled in the art using standard assays (such as described below). Various such compounds are described in published PTC applications WO 96/39384 and WO 96/39385. However, other such glycogen phosphorylase inhibitors will be known to those skilled in the art.

The compounds of formula I and IA may be prepared by methods known in the art. Some processes for the preparation of compounds of formula I and IA, which constitute further features of the present invention, are illustrated below in the reaction schemes.

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Reaction scheme

I

aqueous

OH +

^R 11 ^R 11

Reaction scheme ii ·······················

COOEt

Reaction Scheme III

· · • · • · • • · • • • • • • • · · • • · · · • • • · • · • • • · • · • ···· • · · • • · · · · · • • · ·

Reaction scheme iv

COOH

XXIV

OH

XXVI

COOAlkyl

XXII

R ₁₂ H r ₈ r ₉ n h t> CONR ₈ R _g ; C (O) R ₁₂

Reaction scheme

·· • • • • • • • · · • ··· • · • · • • • · ···· · · • · • · • · · • · • ··· • • • • · · · · · · • • · · ·

R3 xxx

^R 3 XXXII

^R 3 XX

999 ··· ·· • • • · · · · · · · · · · • ···· · · · · 9 • • • · · · · ····· • M 99 ^and n a

R e scheme yj

R4 R4 A base, R3X p _T NH COOH N COOH I XL 1 R3 XLI X H ₂ H 3 COOH I.PhCHO, reduction χ ΗΙΨ ΊΧ3ΟΗ 2. NaCNBH 3 / R ₃ CHO | XLII 3. exhaust. H2, Pd / C r ₃ XXX

re

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

9 9 9 9 9 9 S B · ··, * · · · ···

Reaction Scheme VII

R e c a tic scheme VIII

· · · · · · • ·· • • • • · • rf · • • · · · • · • · * · • • • · • · · ···· · · · • · · · · • e • ··

Pt

LXIII

LXVI ················

Reaction scheme

LXVIII r ₃

Et ₂ NSF ₃

-------- ►

--------- ►

Et ₂ NSF ₃

LXIX

Reaction scheme

According to Reaction Scheme I, compounds of formula I wherein R 1, R _10, R _11f A, R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R _{7 may} be as defined above may be prepared by any of the two following: ··········································· In the general procedures outlined above. In the first process, a compound of formula (I) may be prepared by coupling an appropriate indole-2-carboxylic acid or an indoline-2-carboxylic acid of formula (I) with the corresponding amine of formula (III). The compound of formula

III (i.e. by acylating the amine with a second of these processes I may be prepared by coupling the corresponding compound of formula IV (i.e., a compound of formula I wherein R 8 is carboxy) with an appropriate alcohol of formula R 8 R 8 NH or an amine or alcohol of formula R ₁₂ H R _g, R g, and R ₁₂ are as defined above (i.e., acylating the amine or alcohol).

Typically, a compound of formula II is mixed with a compound of formula III (or a compound of formula IV is mixed with an appropriate amine) (e.g., R ₁₂ H or R 8 R 8 NH) or an alcohol in the presence of a suitable coupling agent. A suitable coupling agent is one which converts a carboxylic acid into a reactive substance that is capable of reacting with an amine or an alcohol to form an amide or ester linkage.

The coupling agent may be a reagent that is capable of causing condensation of these substances in a single reaction vessel when the carboxylic acid is mixed with an amine or an alcohol. If the acid is to be condensed with an alcohol, a large excess of this alcohol is preferably used as the reaction solvent, optionally with the addition of 1.0 to 1.5 equivalents of added dimethylaminopyridine. Examples of coupling agents include the combination of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with hydrobenzotriazole (DEC / HBT), carbonyldiimidazole, a combination of dicyclohexylcarbodiimide with hydroxybenzotriazole (HBT), and

2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), Combination 80 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), Combination 80 Carbonyldiimidazole and HBT and diethylphosphoryl cyanide. The coupling is carried out in an inert solvent, preferably an aprotic solvent, at a temperature of about -20 to about 50 ° C, for a period of about 1 to 48 hours. Examples of suitable solvents include acetonitrile, dichloromethane, dimethylformamide and chloroform.

A coupling agent can also be used to convert the carboxylic acid to an activated intermediate which is isolated and / or formed in the first step, and then the intermediate is reacted in the second step with the appropriate amine or alcohol. Examples of such condensing agents include thionyl chloride and oxalyl chloride (which converts the carboxylic acid to the acid chloride), fluoride (the carboxylic acid is the reagent), or an alkyl chloroformate such as isopropenyl chloroformate (in the presence of cyanuric acid converts to isobutyl or tertiary) amine base) (this reagent converts the carboxylic acid into a mixed carboxylic acid anhydride). When oxalyl chloride is used as the coupling agent, preferably a small amount of dimethylformamide is used as a cosolvent to be added to another solvent (such as dichloromethane) to catalyze the formation of the acid chloride. The use of these coupling agents and the appropriate choice of solvents and temperatures are within the skill of those skilled in the art and can also be readily determined based on literature. These and other exemplary conditions that are suitable for carboxylic acid couplings are described in Houben-Weyl, Vol. XV, Part II, E.Winch, Ed., G.Thieme Verlag, 174, Stuttgart and M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag Berlin, 1984, and The Peptides, Analysis, Synthesis and Biology (ed. E. Gross and J. Meinhofer), Vol. 1 to 5 (Academic Press, NY, USA, 1979 to · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ···· · · · · · · · · · · · · · · · · · · · ·

1983).

The compounds of formula IV, wherein R-PR-Q ^, R _ιχ, A, R _2, R _3, R _4, R _5, and _R? as defined above, may be prepared from the corresponding ester of formula V (i.e., a compound of formula I wherein R 8 is C 1 -C 5 alkoxycarbonyl or benzyloxycarbonyl) by hydrolysis with aqueous alkali at a temperature of about -20 to about 100 ° C, usually at about 20 ^° C, for about 30 minutes to about 24 hours.

Alternatively, the compounds of formula IV are prepared by activating the indocarboxylic acid of formula II with a coupling reagent (described above) and the resulting activated intermediate (such as acid chloride, acid fluoride or mixed anhydride) is then reacted with a compound of formula III wherein R ₃ , R ₄ , R ₅ and R 4? are as defined above and R8 is carboxy, in a suitable solvent, in the presence of a suitable base. Suitable solvents include water, methanol and mixtures thereof, together with a co-solvent such as dichloromethane, tetrahydrofuran or dioxane. Suitable bases include sodium, potassium or lithium hydroxide, sodium or potassium bicarbonate, sodium or potassium carbonate or potassium carbonate together with tetrabutylammonium bromide (1 equivalent) in sufficient quantity to consume the acid liberated in the reaction (usually in an amount such that the pH of the reaction mixture maintained above 8). The base may be added portionwise together with the activated intermediate to ensure appropriate pH control of the reaction mixture. The reaction is usually carried out at a temperature ranging from -20 to 50 ^° C. Isolation procedures can be individually designed by one skilled in the art to remove impurities. Usually, the water-miscible cosolvents are removed

B ·· ·······························

B B ··· · B · · B ·

By evaporation, the impurities are extracted with organic solvents at high pH, acidification to low pH (1-2); and the desired product is extracted with a suitable solvent, filtered off or such as ethyl acetate or dichloromethane.

The compound of formula (V) may be coupled with a suitable alkoxycarbonyl compound using the procedure described above to formula (II) of formula (A) analogously to process (A).

as above, (for example, prepare III, wherein R _{6 is the} compound which is used

Alternatively, the sulfone-containing sulfur atom may be prepared from compounds in the oxidation state of the sulfoxide or the corresponding compounds of formula I containing a sulfur atom in an unoxidized form by treatment with a suitable oxidizing agent such as m-chloroperbenzoic acid in dichloromethane at about 0 to about 0 25 ° C for about 1 to 48 hours (about 1 to about 1.3 equivalents of m-chloroperoxybenzoic acid is required for conversion to a sulfoxide oxidation state, and more than about 2 to be converted to a sulfone oxidation state eq.

The compounds of formula I, which are the amino- moiety in R ₅ mono or dialkylated, it may alternatively be prepared from corresponding compounds of formula I, wherein R ₅ is aminoalkoxy by monoalkylation or dialkylation of the amino group in the residue R ₅ to give the desired compounds of formula I, Such mono- or dialkylation may be carried out by treating the R ₅ -aminoalkoxy compound with one equivalent of a suitable carbonyl compound (in the case of monoalkylation) or with more than two equivalents of a suitable carbonyl compound (in the case of dialkylation) and a suitable reducing agent in the in a suitable solvent. Suitable reducing conditions include the use of sodium cyanoborohydride or sodium borohydride in methanol or ethanol or the use of hydrogen in the presence of a hydrogenation catalyst (such as palladium on carbon) in a polar solvent such as water, methanol or ethanol at about 0 to 60 ^° C for about 1 to 48 hours.

Compounds of formula I wherein R8 is alkanoyloxy (RCOO-) may alternatively be obtained by O-acylation of the corresponding compound of formula I with an appropriate acid chloride or other activated acid derivative, optionally in the presence of a suitable base (e.g. a tertiary amine base such as trialkylamine or pyridine), preferably in an aprotic solvent such as tetrahydrofuran or dichloromethane at a temperature of about 0 to about 50 ^° C for about 0.5 to about 48 hours.

Alternatively, compounds of formula I wherein R8 and Ry together are oxo may be prepared by oxidizing a corresponding compound of formula I, for example, wherein Rg is hydroxy and Ry is hydrogen, with a suitable oxidizing agent. Examples of suitable oxidizing agents include Dess-Martin reagent in dichloromethane, carbodiimide and dimethylsulfoxide and an acid catalyst (Pfitzner-Moffatt conditions or modifications thereof, such as the use of a water-soluble carbodiimide) or a Swern-type reaction (e.g. using oxalyl chloride, dimethylsulfoxide). . Compounds of formula (I) containing other functional groups sensitive to oxidation may be present.

···· protect with suitable protecting groups which are later cleaved off.

For example, some of the compounds of Formula I in Reaction Scheme I contain primary and secondary amino or carboxy groups in the moiety defined by R ₅ or R 8. Which may interfere with the desired coupling reaction of Reaction Scheme I, where the intermediate of formula III or the amine R ₁₂ H ^and L ^e ^ ° RgRgNH left defenseless. During the coupling reaction shown in reaction Scheme I, it may therefore be a primary or secondary amine function is protected by a suitable protecting group, if present in the radicals R ₅ and R g of the intermediate of formula III and the amine (of the formula R _g R _g NH or R ₁₂ H) . As the product of such a coupling reaction, a compound of formula I containing a protecting group is obtained. The present protecting group can be cleaved off in the next step to give a compound of formula I. Suitable protecting groups for protecting the amino and carboxy groups include protecting groups commonly used in peptide synthesis (such as N-tert-butoxycarbonyl, N-benzyloxycarbonylcarbonyl and 9-fluorenylmethyl) in the case of amines and a lower alkyl or benzyl ester group, in the case of carboxylic acids) and which are not chemically reactive under the coupling conditions described above (these conditions are also provided in process A immediately preceding the example) and which can be removed without chemical modification other functional groups in the compound of formula I.

The starting indole-2-carboxylic acid and indoline-2-carboxylic acid used in the reaction depicted in Reaction Scheme I are either commercially available or known in the art (prior art). · · · · · ·

techniques are widely published) or are available by conventional synthetic methods. For example, according to Reaction Scheme II, an indole ester of formula VII can be prepared from a compound of formula VI (wherein Q is selected to yield the residue defined above) by Fischer indole synthesis (see The Fischer Indole Synthesis, Robinson B (Wiley, New York, USA). (1982), followed by saponification of the resulting inol ester of formula VII to give the corresponding acid of formula VIII The starting arylhydrazone can be prepared by condensation of a readily available hydrazine with the appropriate carbonyl derivative or JappKlingeman reaction (see Organic Reactions, Philips RR, 1959, 10, 143 ).

The indole-2-carboxylic acid of formula VIIIA can be prepared by condensation of an o-methylnitro compound of formula IX with an oxalic acid ester, then the indole ester of formula X formed is reduced to a nitro group and the product is hydrolyzed.

This three-step procedure is known as the Reissert indole synthesis (Reissert, Chemische Berichte 1897, 30, 1030). Conditions suitable for carrying out this sequence and related references to further literature are set forth in Kermack, et al., J. Chem. Soc. 1921, 119, 1602; Canon et al., J. Med. Chem. 1981, 24, 238, Julian et al., Heterocyclic Compounds, Vol. 3 (Wiley, New York, USA, R. C. Elderfield, ed.) P. 18. An example of a specific embodiment of this sequence is given in Examples 10A to 10C below.

3-Halo-5-chloro-1H-indole-2-carboxylic acid can also be prepared by halogenating 5-chloro-1H-indole-2-carboxylic acids.

··········································· ·· ·

Alternatively (relative to Reaction Scheme II), substituted indolines of formula (XIV) can be prepared by reducing the corresponding indoles of formula (XV) with a reducing agent such as magnesium in methanol at about 25 ^° C to about 65 ^° C in about 1 to 48 hours. Scheme III).

The indoline carboxylic acids of formula XVI are prepared by saponification of the corresponding ester of formula XVII (see Reaction Scheme III). Compounds of formula XVII are prepared by reducing the corresponding indole esters of formula VII with a reducing agent such as magnesium in methanol as described above for the conversion of compounds of formula XV to compounds of formula XIV.

The following paragraphs describe the preparation of the various amines used in the reactions illustrated in the above schemes.

According to Reaction Scheme IV the Formula XXII compounds (the Formula III amines of Reaction Scheme I wherein R ₅ is hydroxy, _R? Is H and R ₆ is an ester) or Formula XXVI compounds (R _g represents a group of formula C ( O) NR ₈ R ₈ or C (O) R ₁₂ ) are prepared from an N-protected (protected by skupinou) aldehyde of formula XX. The aldehyde XX or the aldehyde adduct XX with sodium bisulfite is treated with potassium cyanide or sodium cyanide in aqueous solution in the presence of a co-solvent such as dioxane or ethyl acetate at a temperature of about 0 to about 50 ° C to form the cyanohydrin of formula XXI . This cyanohydrin is treated with an alcohol (for example, an alcohol (for example). (C 1 -C 6 alkanolol, such as methanol) and a very strong catalyst, such as hydrogen chloride, at a temperature of from about ⁰ ° C to about 50 ^° C, and then water is optionally added to the reaction mixture. If the protecting group P _T is still present, it is cleaved by a suitable deprotection method to give a compound of formula XXII. For example, if the N-protecting group Pip in a compound of formula XX is a t-butoxycarbonyl (t-Boc) group, a compound of formula

XXIII directly from the compound of formula XXI and the addition of water is not required. A compound of formula XXII may be protected on nitrogen with a suitable protecting group to give a compound of formula XXIII. The ester can then be hydrolyzed with aqueous alkali at a temperature of from about 0 ° C to about 50 ° C in a solvent inert to this reaction to give the corresponding hydroxy acid of formula XXIV. Compound of formula

XXIV compound is coupled (in an analogous procedure to the coupling process described in Reaction Scheme I) with the appropriate amino compound of the formula HR RgRgNH or ₁₂ · The compound of formula XXV ,, which arises in the reaction, then deprotected to give the compound of formula XXVI ( i.e., a compound of formula III wherein R ₅ is hydroxy, R ₇ is hydrogen and R 8 is C (O) R ₁₂ or C (O) NR 8 R 8 Example of conversion of cyanohydrin XXI to the corresponding methyl ester XXII with cleavage t-Boc protecting groups are disclosed in Example 1a of PCT publication WO / 9325574. Other examples of reactions in which cyanohydrin is converted to lower alkyl esters of formula XXIII can be found in US Patent No. 4,814,342 and EP 0 438 233.

Some compounds of formula I are stereoisometric due to the stereochemical configuration at the carbon atoms designated by a and b. A person skilled in the art ···························· Can produce intermediates of formula XXII by the desired stereochemistry according to the reaction scheme and XXVI with

IV. For example, the aldehyde of formula XX is available in both enantiomeric forms (stereochemistry of a) by procedures described in the literature below (see Reaction Scheme V). Cyanohydrin of formula XXI may be prepared from a compound of formula XX by treatment with sodium or potassium cyanide as described above, while maintaining the stereochemistry at carbon and. This gives a mixture of stereoisomers on carbon b.

After separation of the isomers or purification of one of the isomers, crystallization can be used at this stage.

Preparation of a compound of formula XXI wherein P _T is BOC, R ₃ is hydrogen, R ₄ is benzo, stereochemistry at carbon a is (S) and at carbon b (R) using the above procedure, as well as recrystallization purification, are described in Biochemistry 1992, 31, 8125-8141.

Alternatively, the separation of isomers may be accomplished by chromatographic or recrystallization techniques following conversion of a compound of formula XXI (mixture of isomers) to a compound of formula XXII, XXIII, XXIV, XXV, XXVI, V, IV or I by procedures and / or process sequences as described in of this description. Intermediates of formula XXI with specific stereochemistry at carbon atoms a and b may be converted to intermediates of formula XXII while maintaining this stereochemistry with an alcohol and a very strong catalyst and optionally adding water as described above by reaction with the following.

································

Alternatively, the desired isomer of the compound of formula XXI may also be obtained by derivatization of the intermediate of formula XXI and chromatographic separation of diastereomeric derivatives (e.g. using trimethylsilyl chloride (TMS) or tert-butyldimethylsilyl chloride (TBDMS) to give O-TMS or O-TBDMS derivatives) . The silyl derivative of the intermediate of formula XXI with a single stereoisomeric form on carbon atoms a and b is converted to the intermediate of formula XXII while maintaining stereochemistry (if the silyl group is not cleaved at this stage, cleaved additionally by a suitable method such as tetrabutylammonium fluoride in tetrahydrofuran) in connection with the conversion of compounds of formula XXI to compounds of formula XXII.

According to Reaction Scheme V, the aldehydes of formula XX (starting material for the process shown in Reaction Scheme IV) are prepared from the corresponding amino acids of formula XXX. The amino acid of formula XXX is protected on nitrogen by a protecting group P _T , such as BOC. The protected compound is esterified with an alcohol and converted to an ester, preferably the methyl or ethyl ester of the compound of formula XXXI. This can be accomplished by reacting a compound of Formula XXX with methyl or ethyl iodide in the presence of a suitable base (e.g. potassium carbonate in a polar solvent such as dimethylformamide. The compound of Formula XXXI is reduced, e.g., diisobutylaluminum hydride in hexane or toluene or mixtures thereof at about 78 to about -50 ° C, followed by quenching the reaction mixture with methanol at -78 ^° C, as described in J. Med. Chem., 1985, 28, 1779-1790, to give the aldehyde of formula XX. shown in Reaction Scheme V), analogous Nmethoxymethylamides (corresponding to the compound of formula

· · · · · · • · · • • • • • • • • · • · • • · · · • • • · • · • • • • · • · • ···· • · · • • · · · · • · • • · · · ·

XXXI, but wherein the alcohol substituent of the ester is replaced with N (OMe) Me) to prepare from the compound of formula XXX, Ν, Ο-dimethylhydroxylamine and a suitable coupling agent (e.g. 1- (3-dimethylaminopropyl) -3-ethylcarbodiimidyl chloride (DEC)). The resulting compound is reduced, for example, with lithium aluminum hydride in a solvent inert to the reaction, such as ether or tetrahydrofuran, at a temperature of from about 0 to about 25 ° C to give the aldehyde of formula XX.This two-step method is generally applicable to conversion. of protected β-amino acids to the aldehydes of formula XX (Fehrentz and Castro, Synthesis 1983, 676-678).

Alternatively, aldehydes of formula XX may be prepared by oxidation of protected amino alcohols of formula XXXIII, for example using pyridine / sulfur trioxide complex, at a temperature of about -10 ^° C to about 40 ^° C in a solvent inert to this reaction, such as preferably dimethylsulfoxide. The protected amino alcohols of formula XXXIII are either commercially available or can be prepared by protecting amino alcohols of formula XXXII. Aminoalcohols of formula XXXII are prepared by reduction of amino acids of formula XXX. This reduction is accomplished by treating compounds of formula XXX with lithium aluminum hydride as described by Dickman et. al., Organic Synthesis, Wiley, New York, 1990, col. Vol. VII, p. 530 or treatment with a mixture of sulfuric acid and sodium borohydride as described in Abiko and Masamune, Tetrahedron Lett. 1992, 233, 5517-5518 or also by treatment with a mixture of sodium borohydride and iodine as described by McKennon and Meyers, J. Org.Chem. 1993, 58, 3568-53571. Finally, reference is made to other suitable procedures for converting amino acids of formula XXX to amino alcohols of formula XXXII.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· ·· ·· · ··

Compounds of formula XXX used in the process shown in Reaction Scheme V can be prepared according to Reaction Scheme VI as follows: First, amino acids of formula XLI are prepared by alkylating protected (P _T ) amino acids of formula XL using a suitable base and an alkylating agent. . Specific alkylation 1977, 55, 945 to procedures suitable for this Benointon, Can.J. Chem. Org. Chem. 1985, 50, methyl, can be used, for example, as indicated

906 to 910

950th

Hansen, J. Presents

When R ₃ sodium hydride and methyl iodide in tetrahydrofuran. The compound of formula XXX is then obtained by cleavage of the protecting group from the compound of formula XLI.

Alternatively, the amino acid of formula XLII can be N-alkylated via a three-step process involving reductive benzylation (e.g., palladium-on-carbon hydrogenation in the presence of benzaldehyde). The obtained mono-N-benzyl derivative is then reductively aminated with a suitable acyl compound (e.g., formaldehyde and sodium cyanoborohydride to introduce the methyl group R ₃ ) to give the N-benzyl-NR ₃ -substituted amino acid. The N-benzyl protecting group is readily cleaved (for example, by hydrogenation in the presence of a suitable catalyst) to give the compound of formula XXX. Specific conditions suitable for this three-step alkylation procedure are described in Reinhold et al., J. Med. Chem. 1968, 11, 258-260.

The preparative procedure described immediately above can also be used to introduce the R ₃ group into an intermediate of formula XLIV to give an intermediate of formula XLV (which is an intermediate of formula III wherein R 6 is hydroxy). Preparative procedure as described. Directly above can also be used to introduce the R ₃ group into an intermediate of formula IIIa (which is an intermediate of formula III wherein R ₃ represents an atom H).

The amino acids used in the procedures of the schemes shown in this specification (e.g., compounds of formulas XL and XLII) are either commercially available, known from the literature, or can be prepared by various methods known to those skilled in the art. For example, Strecker synthesis and variations thereof may be used. Thus, the aldehyde R ₄ CHO can be reacted with sodium or potassium cyanide and ammonium chloride to give the corresponding aminonitrile. This amino nitrile is hydrolyzed with a mineral acid to give the desired amino acid of formula XLII (R ₄ C (NH ₂ ) COOH). Alternatively, the Bucherer-Berg method may be used in which a hydantoin is prepared by heating an aldehyde of formula R ₄ CHO with ammonium carbonate and potassium cyanide and gradually hydrolyzing it (e.g. with barium hydroxide in refluxing dioxane) by acid or base to give the desired amino acid XLII ( r ₄ c (n ₂ ) cooh).

Other methods for synthesizing amino acids allowing one skilled in the art to prepare the desired intermediate amino acids of formula XLII (R ₄ C (NH ₂ ) COOH) required for the synthesis of compounds of formula I are also described in the literature.

Suitable methods of synthesis and resolution to the isomers of compounds of formula XLII can be found in the reviews by Duthaler, Tetrahedron 1994, 50, 1539-1650 or William R.

M. Synthesis of Optically Active Amino Acids, Pergamon, Oxford, UK, 1989.

· · • · • · • ·· · • · • • · • • • · · • • · · · • • • · • • • • • • · • · • ···· • · • • • • • · • • • • • • · · «· • · • · · · · ·

A specific method for synthesizing an intermediate of formula XLII in any of the enantiomeric forms of the corresponding intermediate of formula R ₄ X wherein X is chlorine, bromine or iodine is that of Pirrung and Krishnamurthy (J. Org. Chem. 1993, 58, 957-958), or by following the ^F O'Donnell, et al. (J. Am.

Soc. 1989, 1111 2353-2355). The desired intermediates of formula R ₄ X are readily prepared by a variety of methods known to those skilled in the art. For example, compounds in which R ₄ X represents a radical formylation group of ArCH ₂ X may be prepared by halogenating a compound of formula ArCH ₃ or an arene of formula Ar-H and converting an alcohol to a bromide.

Another specific method for the synthesis of intermediates of formula XLII in any enantiomeric form is that of Corey and Linka (J. Am. Chem. Soc. 1992, 114, 1906-1908). The intermediate of formula R ₄ COCl _{3 is} enantiospecificly reduced to an intermediate of formula R ₄ CH (OH) CCl ₃ , which is converted to an intermediate of formula R ₄ CH (N ₃ ) COOH by treatment with azide and base and the latter compound is reduced by catalytic hydrogenation to the desired a compound of formula XLII. The necessary trichloromethylketone (R ₄ COCCl ₃ ) is obtained by reacting an aldehyde of the formula R ₄ CHO with a trichloromethyl anion followed by oxidation (Gallina and Giorgano, Synthesis 1989, 466-468).

Intermediate amines of general formula III (used in Reaction Scheme I), wherein R ₅ and R _{5 are} R ₅ . each of which is hydrogen, can be prepared as shown in Reaction Scheme 7. The amino acid of formula L (P _T -protected) is activated by conversion to an acid chloride, an acid fluoride or a mixed anhydride (e.g. using iso-butyl chloroformate and triethylamine in an inert solvent, such as tetrahydrofuran or dioxane, at a temperature of about -0 to about -40 ° C), and the activated intermediate is then reacted with diazomethane to form the diazoketone of Formula LI. The diazoketone of formula LI is treated with an alcohol of formula ROH (for example, a C 1 -C 6 alkanol, such as methanol) and a suitable catalyst such as heat, silver oxide or silver benzoate to form the ester of formula LII. The ester of formula LII is deprotected to give a compound of formula IIIA (Wolf rearrangement). Alternatively, the ester of formula LII can be hydrolyzed, for example by alkali, and condensed with a suitable amine of formula R ₁₂ ^H or HNR ₈ R ₈ to give a compound of formula IIIB, as described above.

Intermediate amines of formula III wherein R ₅ is an oxygen-bonded substituent (e.g., alkoxy) (used in Scheme I) may be prepared as shown in Reaction Scheme VIII as follows: a compound of Formula LXI is alkylated on oxygen by treatment with a suitable alkylating agent (e.g. alkyl iodide, alkyl bromide, alkyl chloride, or alkyltholenesulfonate) and a base (sodium hydride or potassium hydride) in an amount sufficient to form the alkoxide, in a suitable polar aprotic solvent (e.g., dimethylformamide or tetrahydrofuran) at a temperature of about 0 to 150 ^° C. of formula LXII. The desired amine intermediate is obtained by deprotection of the compound of Formula LXII.

Intermediate amines of formula III wherein R ₅ is C 1 -C 6 alkoxycarbonylalkoxy (used in Reaction Scheme I) may be prepared as follows: a compound of formula LXI is alkylated with a haloalkanoic acid ester to give a compound of formula LXIII, which is deprotected is converted to the desired amine. The corresponding acid can be prepared

·· • · • · • · · · • ··· • · • · • · • · • · · • • • · ···· ·· · • · • · • · · • · • • · · · · • · • · · · · ·

by hydrolyzing this ester using an aqueous alkali in a suitable solvent. The amines of formula III, wherein _Rg contains an ester and _R5 contains a carboxy may be prepared from the Formula LXIII amine (as prepared above in this paragraph), wherein _R5 contains the carboxylic acid functionality, butyl ester by the action of anhydrous acid to provide the corresponding acid at R ₅ without hydrolyzing the ester at position R _g.

Compounds of formula LXVI (intermediate amines of formula III wherein R 8 is protected aminoalkoxy) can be prepared from a compound of formula LXI. The compound of formula LXI is alkylated with haloalkanenitrile to give a compound of formula LXIV. The compound of formula LXIV is reduced to the primary amine by treatment with hydrogen in the presence of a suitable catalyst (e.g., carbonate) and in the presence of ammonia, preferably in a polar protic solvent such as water, methanol or ethanol, to form the primary amine of formula LXV. The compound of formula LXV is protected on a nitrogen protecting group which is orthogonal to another protecting group, the protecting group P _{T of the} compound of formula III is cleaved. Protected by formula III with ^P i

Drinking and then the desired general to give the compound of formula II of formula i is coupled with the appropriate and the resulting protected deprotected.

compound compound

The compounds of formula LXIII and LXIV, wherein n is 2, are preferably prepared by treating the compound of formula LXI with an excess of an acrylic ester or acrylonitrile in the presence of a suitable base, such as potassium or sodium hydroxide, in a suitable solvent, preferably polar a protic solvent.

♦ · · * * * φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ · · · · · · · · · · · · · · ·

According to Reaction Scheme IX the Formula LXVII compound and LXIX (compound of formula III, wherein _R5 is F or _R5 and _R are both F) may be prepared from the Formula LXI compound. The compound of formula LXI is treated with a suitable fluorinating agent, such as diethylaminosulfurtrifluoride in a solvent inert to this reaction, such as an aprotic solvent, preferably dichloromethane, to form a compound of formula LXVII. Suitably, the compound of formula LXVII is deprotected.

The compound of formula LXI is oxidized to the compound of formula LXVIII using the conditions described above for the preparation of a compound of formula I wherein R ₅ and R 6 are hydrogen. together form an oxo group. The compound of formula LXVIII is difluorinated under appropriate conditions (for example, by treatment with diethylaminosulfur trifluoride in dichloromethane).

According to Reaction Scheme X the Formula LXXIII compound or Formula LXIV, wherein R ₇ is alkyl (i.e., Formula III compound wherein _R? Is alkyl) are prepared from the Formula LXX compound (analogous amine preparation is also described in Reaction Scheme V). The compound of formula LXX is treated with an organometallic reagent of formula R _? M and the resulting secondary alcohol are oxidized as described in the previous paragraph to the compound of formula LXXI. The compound of formula LXXI is converted via a cyanohydrin of formula LXXII to a compound of formula LXXIII using the same conditions as used to convert a compound of formula XXI to a compound of formula XXII (see Reaction Scheme IV).

Alternatively, a compound of Formula LXXII is converted to a compound of Formula LXIV as described for the conversion of a cyano intermediate to an amide according to Reaction Scheme V.

·············································· · ··· ·· ·· ·· · ·· ···

A compound of the formula R _g NH _2, or R _g NH ₂ is monoalkylated with a carbonyl compound corresponding to R _g and R _g under suitable reductive amination conditions, to give the amine of the formula R _g R _g NH. To avoid dialkylation may be preferable to protect the amines (of formula RgNH ₂ or R _g NH ₂₎ with a suitable protecting group P _T to give the protected amine of formula R ₈ (P _T) NH or R _g (P _T) NH, for example by reaction with benzaldehyde and a reducing agent. The protected amines are monoalkylated with a carbonyl compound corresponding to R _g and R _g under suitable reductive amination conditions, to give the amine of formula všeobcného R _g R _g, N (P _T). The protecting group P _T is removed (e.g. by exhaustive catalytic hydrogenation when P _T is benzyl) to give a compound of formula R _g R _g NH. Appropriate reductive amination conditions can be readily determined by one skilled in the art from the literature. These are disclosed in Borch et al., J. Am.

conditions include conditions

Chem. Soc., 1971, 2897-2904 and in Emerson, Organic Reactions, Wiley, New York, 1948 (14), 174, Hutchins et al., Org. Prep. Proced. Int 1979 (11), 20 and Lane et al., Synthesis 1975, 135. Conditions for reductive amination favoring N-monoalkylation are described, for example, in Morales et al., Syntetic Communications 1984, 1213-1220 and Verado et al., Synthesis, 1992, 121 to 125. the amines of the formula R _g NH _2, or R _g NH ₂ may also be monoalkylated by the appropriate compound of formula R _g R _g X and X, wherein X is chloride, bromide, methanesulfonate or tosulfonátový. Alternatively, an intermediate of formula R _g (P _T ) NH or R _g (P _T ) NH may be alkylated with an appropriate compound of formula R _g X or R _g X and the protecting group may be cleaved to give a compound of formula R _g R _g NH.

For the preparation of amines of the formula R _g R _g NH, wherein R _g, R _g NH or NH bonded oxygen-nitrogen, may be used other methods. Thus, the readily available compound may be an alkoxycarbonyl-NHOH formula. (with 1 to 4 carbon atoms in the alkoxy moiety) or NH ₂ CONHOH dialkylated on nitrogen and oxygen with a base and an excess of a suitable alkylating agent of formula RX to form the corresponding compound of formula alkoxycarbonyl-N (R) OR (1 to 4 carbon atoms in alkoxy) part) which is then hydrolyzed to give a compound of formula R ₈ R _g NH (wherein R _g = R _g = R). Suitable conditions, bases and alkylating agents are described, for example, in Goel and Krolls, Org.Proced.Int. 1987, 19, 75-78 and Major and Fleck, J. Am. Soc. 1928, 50, 1479. Alternatively, the amine of formula

NH ₂ CONH (OH) alkylated sequentially, first on oxygen, to form a compound of formula NH ₂ CONH (OR *) and then on nitrogen, to form a compound of formula NH ₂ CON (R) (OR *) by sequential treatment with an alkylating agent of formula R * X and RX in the presence of a suitable base. Suitable bases and alkylating agents are disclosed in Kreuzkamp and Messinger, Chem. Ber. 100, 3463-3465 (1967) and Danen et al. (J. Am. Chem. Soc. 1973, 95, 5716-5724). Hydrolysis of these alkylated hydroxyurea derivatives yields the amines of formula R'ONH ₂ and R'ONHR, which correspond to certain formula R _g amine R _g NH. Those skilled in the art may adapt the procedures described in this paragraph to include other alkylating agents, R, R ', and other alkylating agents.

RX to prepare other amines of formula R _g R _g NH wherein the groups R _g and R _g N-N are connected to oxygen-nitrogen linked. Uno et al.

(SynLett 1991, 559-560 describe the addition of an organometallic reagent of the formula I R ¹ CH = N-OR ¹¹ which is catalyzed

R-Li to O-alkyloxime of the formula boron trifluoride to form compounds of formula R'RCH-NH (OR). This method can also be used to obtain compounds of formula

R _g R _g NH, wherein one of the radicals R _g -NH and R _g -NH is attached by an oxygen-nitrogen bond.

Prodrugs of the invention in which it is carboxyl

· · · · • · • · · • • • • • • • • • · • • · · · • • • · • • • • • · • · • ···· • · • • · · · · • · • • · • ·

a carboxylic acid group of the formula I replaced by an ester group may be prepared by treating the carboxylic acid with a suitable alkyl halide in the presence of a base such as potassium carbonate in an inert solvent such as dimethylformamide at a temperature of about 0 to 100 ^° C for about 1 to about 24 hours. Alternatively, the acid may be mixed with an appropriate alcohol as a solvent in the presence of a catalytic amount of an acid, such as concentrated sulfuric acid, at a temperature in the range of about 20 to about 120 ° C, preferably at reflux. 1 to 24 hours. Another way of carrying out this reaction is by mixing the acid in the presence of a solvent, formed in a process with a stoichiometric amount of a catalytic amount of tetrahydrofuran while continuously removing by physical use of a Dean-Stark trap. for example using molecular sieves).

Prodrugs of the invention in which an alcohol functional group is derivatized to form an ether can be prepared by mixing the alcohol with a suitable alkyl bromide or alkyl iodide in the presence of a base such as potassium carbonate in an inert solvent such as dimethylformamide and allowing the reaction to proceed. at a temperature in the range of about 0 to about 100 ° C for about 1 to 24 hours. Alkanoylaminomethyl ethers can be obtained by reacting an alcohol with bis (alkanoylaminomethane) in the presence of a catalytic amount of an acid in an inert solvent such as tetrahydrofuran, as described in US 4,997,984. Alternatively, these compounds can be prepared by the methods described in Hoffman et al. 1994, 59, 3530.

100 ····························

Dialkyl phosphate esters can be prepared by reacting an alcohol with a dialkyl chlorophosphate in the presence of a base in an inert solvent such as tetrahydrofuran. Dihydrogen phosphates may be prepared by reacting an alcohol with diaryl or dibenzyl chlorophosphate as described above followed by hydrolysis or hydrogenation in the presence of a noble metal catalyst.

Glycosides can be prepared by reacting an alcohol with a carbohydrate in an inert solvent such as toluene in the presence of an acid. The water produced in the reaction is usually continuously removed as described above. Alternatively, the reaction may be carried out by treating the alcohol with a suitably protected glycosyl halide in the presence of a base followed by cleavage of the protecting group.

N- (1-hydroxyalkyl) amides, N- (1-hydroxy-1- (alkoxycarbonyl) methyl) amides or compounds wherein the residue R ₂ is replaced by a radical of formula C (OH) C (O) OY may be prepared by reaction of a parent amide or indole with the corresponding aldehyde under neutral or basic conditions (e.g. in the presence of sodium ethoxide in ethanol) at a temperature ranging from 25 to 70 ° C. N-Alkoxymethylindoles or N1- (alkoxy) alkylindoles can be prepared by reacting an N-unsubstituted indole with the desired alkylhaxlogenide in the presence of a base in an inert solvent. 1- (N, N-Dialkylaminomethyl) indole, 1- (1N, N-dialkylamino) ethyl) indole and N, N-dialkylaminomethylamides (e.g. compounds wherein R ₃ is dimethylaminomethyl) may be prepared by reacting the parent NH-compound with the appropriate aldehyde and an amine in an alcoholic solvent at 25 to 70 ° C.

Cyclic prodrugs (e.g., prodrugs of the invention,

101 · e e e e e e e e e e e e e e e e e e e e e e e e e e In which R ₂ and R ₃ represent a common carbon atom, may be prepared by reacting the parent compound (drug) with an aldehyde or ketone or its dimethylacetal in an inert solvent in the presence of a catalytic amount of acid while removing water or methanol. Alternatively, these compounds can be prepared by reacting an amino alcohol or hydroxiamide with a gem-dibromoalkane in the presence of a base (e.g. potassium carbonate) in an inert solvent (e.g. dimethylformamide).

Compounds of formula IA can be prepared by the methods described below.

The numbering of the reaction schemes and formulas given in the following section is independent of the numbering in the previous text (thus, the numbering used in the preceding text should not be confused).

102

REACTION SCHEME XI

······························

R10 R11

105

REACTIVE WITH CH M M XII XII · · · · · · · • • · XII XII XII XII XII XII XII XII · · · · · · ·· ···

COOEt

COOEt COOH

R10 Rn R10 R11

COOEt 1. (ROCO2, ^base / /, nh * Ri-lf- 2. reduction conditions x

R10 R11 hydrolysis conditions

R10 R11

104 • · ·

Reaction scheme

XIII

105

Reaction Scheme XIV

XVIII

COOH

R 12 H R ₈ R ₉ NH

conr ₈ r ₉ ; C (O) R 12 xxv

conr ₈ r ₉ : C (O) R 12

IIIb

106

R e a g g scheme XV

· · · · · · • ·· 9 • • • • • • • · · · • • · · · • • • · • · • • • • · • · • ···· · • · • • · · • · · · • · · ·· ·

COOR

COOH

COOR

XXXIII

107 Reaction Scheme XVI

COOH

COOH

XL

XLI

1. PhCHO, reduction

2. NaCNBH 3 * suitable carbonyl compound

COOH

XXX

3. exhaust ^ 2 <Pd / C

-------- ►

According to Reaction Scheme XI, compounds of formula IA wherein R 1 ^{, R} 10 ^{9 R} 11 ^R 2 ^{9 R} 3 ^{9 R} 4 ^{9 R} 5 ^and R 8 are as defined above may be prepared by any of the following two general procedures. In the first process, a compound of formula IA can be prepared by coupling an appropriate indole-2-carboxylic acid, indoline-2-carboxylic acid or benzimidazole-2-carboxylic acid of formula II with the corresponding amine of formula III (ie acylating the amine of formula III) . In the second process the desired Formula IA may be prepared by coupling the appropriate Formula IV compound (i.e. a compound of formula IA, wherein R is carboxy) with the appropriate alcohol or amine of formula RGR _{g of} NH or R ₁₂ H, wherein R, R _g and R ₁₂ are as defined above (i.e., by acylation of this amine or alcohol). The first procedure (copulation

108 ···························· · 9 · · · · ··· A compound of formula (II) with a compound of formula (III) is generally preferred if R _{4 is} not hydrogen and R ₅ is hydrogen.

Typically, a compound of formula II is mixed with a compound of formula III (or a compound of formula IV is mixed with an appropriate amine (e.g. ^R 12 ^H or R ₈ R ₉ NH)) or an alcohol in the presence of a suitable coupling agent. A suitable coupling agent is one which converts a carboxylic acid into a reactive substance that is capable of reacting with an amine or an alcohol to form an amide or ester linkage.

The coupling agent may be a reagent that is capable of inducing a one-stage condensation of these compounds upon mixing the carboxylic acid with an amine or an alcohol. If the acid is to be condensed with an alcohol, a large excess of this alcohol is preferably used as the reaction solvent, optionally with the addition of 1.0 to 1.5 equivalents of added dimethylaminopyridine. Examples of coupling agents include a combination of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with hydroxybenzotriazole (DEC / HBT), carbonyldiimidazole, a combination of dicyclohexylcarbodiimide with hydroxybenzotriazole (HBT), and 2-ethoxy-1-ethoxy-1-ethoxy-1-ethoxy-1-ethoxy (EEDQ), a combination of carbonyldiimidazole and HBT, propanophosphonic anhydride (PPA), and diethylphosphoryl cyanide. The coupling is carried out in an inert solvent, preferably an aprotic solvent, at a temperature of about -20 to about 50 ° C, about 1 to about 48 hours, and optionally in the presence of a tertiary amine base such as triethylamine. Examples of suitable solvents include acetonitrile, ethyl acetate, dichloromethane, dimethylformamide and chloroform, and mixtures thereof. As an example of a suitable coupling

109 ···············

· · • · · • • • • • • • • · • · • t • ···· • · • • • • • • · · • · ·

The process A mentioned in this description just before the examples of implementation can be mentioned.

A coupling agent which also converts a carboxylic acid to an activated intermediate that is isolated and / or formed in the first step can also be used. Thereafter, this intermediate is reacted in the second step with the appropriate amine or alcohol. Examples of such condensing agents include thionyl chloride and oxalyl chloride (converting the carboxylic acid to an acid chloride), cynauric acid fluoride (converting the carboxylic acid into an acid fluoride), or an alkyl chloroformate such as isobutyl or isopropenyl chloroformate (in the presence of an amine terformate). base (this reagent converts the carboxylic acid into a mixed carboxylic anhydride). When oxalyl chloride is used as the coupling agent, preferably a small amount of dimethylformamide is used as a cosolvent to be added to another solvent (such as dichloromethane) to catalyze the formation of the acid chloride. This acid chloride can be coupled by mixing with an intermediate of formula III in a suitable solvent together with a suitable base. Suitable solvent / base combinations include, for example, dichloromethane, dimethylformamide or acetonitrile or mixtures thereof in the presence of a tertiary amine base such as triethylamine. Other suitable solvent / base combinations include water or C 1 -C 5 alcohols or mixtures thereof together with a co-solvent such as dichloromethane, tetrahydro furan or dioxane and a base such as sodium or potassium carbonate, sodium, potassium or sodium or potassium carbonate. lithium or sodium bicarbonate. The base is used in an amount sufficient to capture the acid released during the reaction. When a mixture of co-solvents is used which are only partially

110 miscible (e.g. dichloromethane / water or dichloromethane / methanol), it is preferred to use a phase transfer catalyst (usually 1-10 mol%), such as a quaternary ammonium halide (e.g. tetrabutylammonium bromide or methyltrioctylammonium chloride). The use of these coupling agents and the appropriate choice of solvents and temperatures are within the skill of those skilled in the art and can also be readily determined based on literature. These and other exemplary conditions that are suitable for coupling carboxylic acids are described in Houben-Weyl, Vol. XV, Part II, E.Winsch, Ed., G. Thieme Verlag, 1974, Stuttgart and M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag Berlin, 1984, and The Peptides, Analysis, Synthesis and Biology (ed. E. Gross and J. Meinhofer), Vol. 1-5 (Academic Press, NY, USA, 1979-1983).

Compounds of formula IV wherein R 1, R ₁₀ R 4 / A, R ₂ , R ₃ , R ₄ and R ₅ are as defined above may be prepared from the corresponding ester of formula (i.e., a compound of formula I wherein R ₆ is alkoxycarbonyl) (1 to 5 carbon atoms in the alkoxy moiety or benzyloxycarbonyl) by hydrolysis with aqueous alkali at a temperature of about -

20 to about 100 ° C, usually at about 20 ° C, about 20 ° C from 30 minutes to about 24 hours.

Alternatively, the compounds of formula IV are prepared by activating the indole carboxylic acid of formula II with a coupling reagent (described above) and the resulting activated intermediate (such as acid chloride, acid fluoride or mixed anhydride) is then reacted with a compound of formula III wherein R _3, R ₄ and R ₅ are as defined above and R _g is carboxy, in a suitable solvent in the presence of a suitable base. Than

111 ······················ Suitable solvents include water, methanol and mixtures thereof, together with a cosolvent such as dichloromethane, tetrahydrofuran or dioxane. Suitable bases include sodium, potassium or lithium hydroxide, sodium or potassium bicarbonate, sodium or potassium carbonate or potassium carbonate with tetrabutylammonium bromide (1 equivalent) in sufficient quantity to consume the acid liberated in the reaction (usually in an amount such that the pH of the reaction mixture maintained above 8). The base may be added portionwise together with the activated intermediate to ensure appropriate pH control of the reaction mixture. The reaction is usually carried out at a temperature ranging from -20 to 50 ° C. Isolation procedures can be individually designed by one skilled in the art to remove impurities. Usually, the water-miscible co-solvents are removed by evaporation, the impurities are extracted with organic solvents at high pH, then acidified to a low pH (1-2) and the desired product is filtered or extracted with a suitable solvent such as ethyl acetate or dichloromethane. .

The compound of formula V may be prepared by coupling the appropriate Formula III, wherein _Rg is alkoxycarbonyl and the appropriate Formula II compound using a procedure analogous to that given above for (napráklad using procedure A).

Alternatively, compounds of formula IA containing a sulfoxide or sulfone oxidation state may be prepared from the corresponding compounds of formula IA containing sulfur in unoxidized form by treatment with a suitable oxidizing agent such as m-chloroperoxybenzoic acid in dichloromethane at a temperature of about 0 to about 25 ^{° C.} C in about 1 to about 48 hours (per conversion)

For a sulfoxide oxidation state, about 1 to about 0.1 is required

1.3 equivalents of m-chloroperoxybenzoic acid, and to be converted to the sulfonic oxidation state, it is necessary to use it in an amount greater than about 2 equivalents.

For example, some of the compounds of formula IA in Reaction Scheme XI contain primary and secondary amino groups and the carboxy groups of the molecule defined by R _g. Which may interfere with the intended coupling reaction of Reaction Scheme XI, if the intermediate of the formula III or an amine of formula R 1 H or R _{2 g,} R ₉ NH left unprotected. During the coupling reaction of Reaction Scheme XI may therefore be a primary or secondary amine function or carboxylic acid function is protected by an appropriate protecting group, if present in the radical R of the intermediate of formula III and the amine (of the formula RGR _g NH or R ₁₂ H). As the product of such a coupling reaction, a compound of formula IA containing a protecting group is obtained. The present protecting group may be cleaved in the next step to give a compound of formula IA. Suitable protecting groups for the protection of amino and carboxy include protecting groups commonly used in peptide synthesis (such as N-tert-butoxycarbonyl, N-benzyloxycarbonyl and 9-fluorophenylmethyleneoxycarbonyl, in the case of amines and the lower alkyl ester carboxy) and which are chemically reactive under the coupling conditions described above (these conditions are also provided in process A immediately preceding the example section) and which can be removed without chemically altering other functional groups in the compound of formula IA.

113 ························ · 9 · ·· ·

The starting indole-2-carboxylic acid and indoline-2-carboxylic acid used in the reaction shown in Reaction Scheme XI are either commercially available or known in the art (the prior art is widely published) or available by conventional synthetic methods. For example, according to Reaction Scheme XII, an indole ester of formula VII (where A is not a nitrogen atom) can be prepared from a compound of formula VI (where Q is selected to yield a residue A as defined above except nitrogen) by Fischer indole synthesis (see The Fischer Indole Synthesis, Robinson B (Wiley, New York, USA, 1982), followed by saponification of the resulting indole ester of formula VII to give the corresponding acid of formula VIII The starting arylhydrazone can be prepared by condensation of a readily available hydrazine with the appropriate carbonyl derivative or Japp. -Klingeman reaction (see Organic Reactions, Philips RR, 1959, 10, 143).

Alternatively, the indole-2-carboxylic acid of formula VIIIA can be prepared by condensation of an o-methylnitro compound of formula IX with an oxalic ester, then the indole ester of formula X is reduced to a nitro group and the product is hydrolyzed.

This three-step process is known as the Reissert indole synthesis (Reissert, Chemische Berichte 1897, 30,

1030). Conditions suitable for carrying out this sequence and corresponding references to further literature are set forth in Kermack, et al., J. Chem. Soc. 1921, 1919, 1602, Cannon et. al., Med. Chem. 1981, 24,238 Julian et. al., Heterocyclic Compounds, Vol. 3 (Wiley, New York, USA, 1962, R.C. Elderfield, ed.) P. 18. An example of a specific embodiment of this sequence is given in Examples 10A to 10C below.

114 9 9

99 ·· · · ··· · ···

3-Halo-5-chloro-1H-indole-2-carboxylic acid can also be prepared by halogenating 5-chloro-1H-indole-2-carboxylic acids.

According to Reaction Scheme XIII, an intermediate benzimidazole-2-carboxylic acid of formula XI can be prepared by condensing an o-diamine compound of formula XIII with a glycolic acid followed by oxidation of the resulting benzimidazole-2-methanol of formula XII (Bistrzycki, A, and Przeworski, G. 1912, 45, 3483).

Alternatively (relative to Reaction Scheme XII), substituted indolines of formula (XIV) may be prepared by reducing the corresponding indoles of formula (XV) with a reducing agent such as magnesium in methanol at a temperature of about 25 to about 65 ° C in 1 to 48 hours. Scheme III).

The indoline carboxylic acids of formula XVI are prepared by saponification of the corresponding ester of formula XVII (see Reaction Scheme XIII). Compounds of formula XVII are prepared by reducing the corresponding indolestees of formula VII with a reducing agent, such as magnesium in methanol, as described above for the conversion of compounds of formula XV to compounds of formula XIV.

The following paragraphs describe the preparation of the various amines used in the reactions illustrated in the above reaction schemes.

According to Reaction Scheme XIV, the N-amino acid of formula XXIII can be protected on nitrogen with a suitable protecting group (P _t ) (e.g., t-butoxycarbonyl - t-Boc) to give a compound of formula XXIV. Those of ordinary skill in the art can readily determine the appropriate protecting group and method thereof

115

· · · · · · • ·· · e · • • · • • · ·· • · · · · • · • · • · * • · • · • · · ···· • · · · • · • · • · • • · · · · · · · · • ·· ···

introduction. For example, the t-Boc protecting group may be introduced by treating the amino acid with a diter. butyldicarbonate, preferably in a protic solvent or a mixture thereof at high pH. Another example of a conventional protecting group is Cbz. This group is introduced by treating the amino acid with benzyl chloride in a suitable, preferably protic solvent or solvent mixture in the presence of a base. The compound of formula XXIV is coupled (by a similar procedure to that described in connection with Reaction Scheme XI) with the corresponding amine of formula

RgRgNH or HR ^{_12, and} the resulting XXV is then de-protecting the compound of formula to form a compound of formula IIIb (i.e. a compound of formula III, wherein

R 8 is C (O) R ₁₂ or C (O) NR 8 R 8).

When the protecting group is a t-Boc group, deprotection of the compound of Formula XXV is accomplished by treatment with an acid in a suitable, preferably aprotic solvent. Among the acids which are suitable for this deprotection are hydrochloric acid, methanesulfonic acid or trifluoroacetic acid.

According to Reaction Scheme XV, a compound of formula XXXI (an N-protected amine of formula III wherein R 8 is C 1 -C 8 alkoxycarbonyl or benzyloxycarbonyl) may be prepared. An N-protecting group (to form a protected amino acid of formula XXXI) is introduced into the unprotected amino acid of formula XXX and then esterified. For example, the esterification of the compound of formula XXXIII can be carried out with a suitable alcohol in the presence of an acid catalyst such as hydrogen chloride or thionyl chloride. When the esterifying agent is to be tert-butanol, the amino acid is reacted with isobutyl and an acid catalyst such as concentrated sulfuric acid. Reaction with an alkyl halide (e.g.

116 (with methyl iodide) and a base (for example potassium carbonate). Alternatively, the esterification may be carried out before the deprotection step.

The Formula XXX compounds wherein _R3 is not H, which are used in the process illustrated in Reaction Scheme X, can be prepared according to Reaction Scheme XVI the following manner: First, the prepared amino acid of formula XLI N-alkylating a protected (P _T) amino acids of the formula XL using a suitable base and an alkylating agent. Specific procedures suitable for this alkylation are described in Benoiton, Can. J. Chem. 1977, 55, 906-910 and Hansen, J. Org.Chem. 1985, 50, 945-950. When R ₃ is methyl and P _T is Boe, for example, sodium hydride and methyl iodide in tetrahydrofuran may be used. The compound of formula XXX is then obtained by cleavage of the protecting group from the compound of formula XLI.

Alternatively, the amino acid of formula XLII can be N-alkylated by a three-step process involving reductive benzylation (for example, by reaction with benzaldehyde and palladium-on-carbon catalyzed hydrogenation). The obtained mono-N-benzyl derivative is then reductively aminated with a suitable carbonyl compound (e.g., formaldehyde and sodium cyanoborohydride, to introduce a methyl group having the meaning of R ₃ ) to give an N-benzyl-NR ₃ -substituted amino acid. The N-benzyl protecting group is readily cleaved (for example, by hydrogenation in the presence of a suitable catalyst) to give a compound of formula XXX. Specific conditions suitable for this three-step alkylation procedure are described in Reinhold et al., J. Med. Chem. 1968, 11, 258-260.

117

The preparative procedure described immediately above can also be used to introduce the general formula IIIa (which is a group R ₃ into an intermediate of formula III wherein R ₃ represents a hydrogen atom).

The amino acids used in the schemes shown in this specification, for example, compounds of formulas XL and XLII are either commercially available, known from the literature, or can be prepared by various methods known to those skilled in the art. For example, Strecker synthesis and variations thereof may be used. Thus, the aldehyde R ₄ CHO can be reacted with sodium or potassium cyanide and ammonium chloride to give the corresponding aminonitrile. This aminonitrile is hydrolyzed with a mineral acid to give the desired amino acid of formula XLII (R ₄ C (NH ₂ ) COOH). Alternatively, the Bucherer-Berg method may be used in which the hydantoin is prepared by heating an aldehyde R ₄ CHO with ammonium carbonate and potassium cyanide; this is gradually hydrolyzed (e.g., barium hydroxide in refluxing dioxane) by treatment with an acid or a base to give the desired amino acid of formula XLII (R ₄ C (NH ₂ ) COOH).

Other methods for synthesizing N-amino acids allowing one of skill in the art to prepare the desired intermadial amino acids of formula XLII (R ₄ C (NH ₂ ) COOH) required for the synthesis of compounds of formula I are also described in the literature.

Suitable methods for the synthesis and / or resolution of the isomers of compounds of formula XLII can be found in the reviews by Duthaler, Tetrahedron 1994, 50, 1539-1650 or Williams R.M., Synthesis of Optically Active Amino Acids, Pergamon, Oxford, UK, 1989.

118 • · ♦

A specific method for synthesizing an intermediate of formula XLII in any of the enantiomeric forms of the corresponding intermediate of formula R ₄ X wherein X is chlorine, bromine or iodine is the method of Pirrung and Krishnamurty (J. Org. Chem. 1993, 58, 957). to 958) or the method of O'Donell et. al. (J. Chem. Soc. 1989, 111, 2353-2355). The desired intermediates of formula R ₄ X are readily prepared by a variety of methods known to those skilled in the art. For example, compounds wherein R ₄ X is ArCH ₂ X may be prepared by radical halogenation of a compound of formula ArCH-j or formylation of an arene of formula Ar-H and conversion of an alcohol to a bromide.

Another specific method for synthesizing intermediates of formula XLII in any enantiomeric form is the Corey and Link method (J. Am. Chem. Soc. 1992, 114, 1906-1908). In this process, the intermediate of formula R ₄ COCCl _{3 is} enantiospecificly reduced to an intermediate of formula R ₄ CH (OH) CCl ₃ , which is converted to an intermediate of formula ₄ CH (N ₃ ) COOH by treatment with azide and base and the latter compound is catalytically hydrogenated to the desired compound. of formula XLII. The necessary trichloromethylketone (R ₄ COCCl ₃ ) is obtained by reacting an aldehyde of the formula R ₄ CHO with a trichloromethyl anion followed by oxidation (Gallina and Giordano, Synthesis, 1989, 466-468).

A compound of the formula R _g NH _2, or R _g NH ₂ is monoalkylated with a carbonyl compound corresponding to R _g and R _g under suitable reductive amination conditions, to give the amine of the formula R _g R _g NH. To avoid dialkylation, it may be preferable to protect the amines (of formula R _g NH _2, or R _g NH ₂₎ with a suitable protecting group P _T to give the protected amine of formula R (Piy) NH or R _g (P _T) NH, for example by reaction with benzaldehyde

119 and a reducing agent. The protected amines are monoalkylated with a carbonyl compound corresponding to Rg or Rg under appropriate reductive amination conditions to provide an amine of the formula RgRgN (Prp). The P _T protecting group is cleaved (for example by exhaustive catalytic hydrogenation when P _T is benzyl) to give a compound of formula R 8 R 8 NH. Appropriate reductive amination conditions can be readily determined by one skilled in the art from the literature. These conditions include those set forth in Borch et. al., J. Am. Chem. Soc., 1971, 2897-2904 and in Emerson, Organic Reactions, Wiley, New York, 1948 (14), 174, Hutchins et al. , Org. Prep. Proced. Int 1979 (11), 20 and Lane et al., Synthesis 1975, 135. Conditions for reductive amination favoring N-monoalkylations are described, for example, in Morales et al., Syntetic Communications 1984, 1213-1220 and Verado et al., Synthesis, et al. 1992, 121 to 125. the amines of the formula R _g NH _2, or R _g NH ₂ may also be monoalkylated by the appropriate compound of formula RGX or RG X, wherein X is chloride, bromide, tosylate or mesylate. Alternatively, an intermediate of formula R 9 (P _T ) NH or R ₉ (P _T ) NH may be alkylated with an appropriate compound of formula R ₉ X or R ₉ X and the protecting group may be cleaved to give a compound of formula R 9 R 9 NH.

Additional methods may be used to prepare amines of the general formula RgRgNH, wherein the Rg-NH or Rg-NH group is bonded by an oxygen-nitrogen bond. Thus, a readily available compound of formula alkoxycarbonyl-NHOH (having 1 to 4 carbon atoms in the alkoxy moiety) or NH ₂ CONHOH can be remotely deprotected on nitrogen and oxygen by treatment with an excess of a suitable alkylating agent of formula RX to form the corresponding compound of formula alkoxycarbonyl -N (R) OR (having 1 to 4 carbon atoms in the alkoxy moiety), which is then hydrolyzed to form a C 1 -C 4 alkoxy group

120 ·

• · · · · · · · · • • · · · · · · ······ • • · · · · · · · · · RgRgNH a compound of formula (wherein R = _Rg = R). Suitable conditions, bases and alkylating agents are described, for example, in Goel and Krolls, Org. Prep. Proced. Int. 1987, 19,

J. Am. Chem.

N-Hydroxyurea first on oxygen, and then on nitrogen, by successive RX of up to 78 and Major and Fleck. Alternatively, it can be alkylated sequentially, of formula NH ₂ CONH (OR ') of formula NH ₂ CON (R) (OR) X a Suitable bases and alkylating agents Kreuzkamp and Messinger, Chem. Ber. Danen et al. (J. Am. Chem. Soc.

SOC. 1928, 50, 1479. The formula NH ₂ CONH (OH) to give the compound to give the compound by treatment with the alkylating presence of a suitable base are disclosed in publications 100, 3463-3465 (1967) and 1973, 95, 5716-5774.

Hydrolysis of these alkylated hydroxyurea derivatives yields the amines of formula R'ONH ₂ and R'ONHR, which correspond to certain amine RGR _{g of} NH. Those skilled in the art can adapt the procedures described in this paragraph, so that it is possible to use other alkylating agents R, R and R -X to prepare other amines of formula RgRgNH, wherein the radicals R, or _{N-Rg-N-linked} are attached oxygen-nitrogen. Uno et al. (SynLett

1991, 559-560) disclose the addition of formula R-Li to an O-alkyloxide of the formula catalyzed by boron trifluoride, an organometallic reagent R'CH = N-OR which is to form compounds of formula R'RCH-NH (OR). The method may also be used to obtain the compounds of formula RgRgNH, wherein one of the radicals of formula R _g R _g NH and NH are oxygen - nitrogen.

Prodrugs of the invention wherein the carboxylic acid group of formula (IA) is replaced by an ester group may be prepared by treating the carboxylic acid group with a suitable alkyl halide in the presence of a base such as potassium carbonate in an inert solvent such as dimethylformamide at about 0 about 100 ^° C for about 1 to 24 hours. Alternatively, the acid may be mixed with an appropriate alcohol such as

121 ··························· A solvent, in the presence of a catalytic amount of an acid such as concentrated sulfuric acid, at a temperature in the range of about 20 ° C to about 120 ° C, preferably at reflux temperature, the reaction time being in the range of about 1 to 24 ° C. hours. Another method of carrying out this reaction is based on mixing the acid with a stoichiometric amount of alcohol in the presence of a catalytic amount of acid in an inert solvent such as tetrahydrofuran, wherein the water formed in the reaction is continuously removed by physical means (e.g. using a Dean-Stark trap) or chemically ( for example using molecular sieves).

Prodrugs of the invention in which an alcohol functional group is derivatized to form an ether can be prepared by mixing the alcohol with a suitable alkyl bromide or alkyl iodide in the presence of a base such as potassium carbonate in an inert solvent such as dimethylformamide and allowing the reaction to proceed. at a temperature in the range of about 0 to about 100 ° C for about 1 to 48 hours. Alkanoylaminomethyl ethers may be obtained by reacting an alcohol with bis (alkanoylamino) -methane in the presence of a catalytic amount of an acid in an inert solvent such as tetrahydrofuran, as described in US 4,997,984. Chem. 1994, 59, 3530.

The dialkyl phosphate esters can be added by reacting the alcohol with dialkyl chlorophosphate in the presence of a base in an inert solvent such as tetrahydrofuran. Dihydrogen phosphates may be prepared by reacting an alcohol with diaryl or dibenzyl chlorophosphate as described above followed by hydrolysis or hydrogenation in the presence of a noble metal catalyst.

122

Glycosides can be prepared by reacting an alcohol with a carbohydrate in an inert solvent such as toluene in the presence of an acid. The water resulting from the reaction is usually continuously removed as described above. An alternative embodiment of the reaction is based on the treatment of the alcohol with a suitably protected glycosyl halide in the presence of a base and subsequent cleavage of the protecting group.

N- (1-hydroxyalkyl) amides, N- (1-hydroxy-1- (alkoxycarbonyl) methyl) amides or compounds wherein the residue R ₂ is replaced by a radical of formula C (OH) C (O) OY may be prepared by reaction of a parent amide or indole with the corresponding aldehyde under neutral or basic conditions (e.g., in the presence of sodium ethoxide in ethanol) at a temperature ranging from 25 to 70 ^° C. N-Alkoxymethylindoles or N1- (alkoxy) alkylindoles may be prepared by reacting N-unsubstituted indoles with the desired alkyl halide in the presence of a base in an inert solvent. 1- (n, N-Dialkylaminomethyl) indoles, 1- (1- (N, N-dialkylamino) ethyl) indoles and N, N-dialkylaminomethylamides (e.g., compounds wherein R ₃ is dimethylaminomethyl) may be prepared by reacting the parent NH-compound with with an appropriate aldehyde and an amine in an alcoholic solvent at 25 to 70 ^° C.

Prodrugs of the invention wherein R ₂ and R ₃ represent a common carbon atom can be prepared by reacting the parent compound (drug) with benzaldehyde or ketone or dimethylacetal in an inert solvent in the presence of a catalytic amount of acid while removing water in methanol.

Starting materials and reagents for the above described

123 ·············· 9,99

9,999 9 9 9 9 9 99

9 9 9 9 9 9 9999 9 9 99

9 · 9 · 9 · 9

Reaction schemes (e.g., amines, substituted indolecarboxylic acids, substituted indolinecarboxylic acids, and amino acids) are also readily available, or can be readily synthesized by conventional organic synthesis methods known to those skilled in the art. For example, many of the intermediates used in the above-described processes for making compounds of Formula IA are related to natural amino acids or are derivatives thereof. Substances of this type are of considerable scientific interest and there is a high commercial demand, and therefore many of these intermediates are commercially available or described in the literature or can be readily prepared from other commercially available substances by methods cited in the literature. Such intermediates contain, for example, compounds of formulas XXX, XLII, XXXII and XXXIII.

Some preparative methods that are useful for preparing the compounds described herein (e.g., compounds of Formula I, compounds of Formula IA) may require the protection of remote functional groups (i.e., primary and secondary amino groups, and also carboxy groups) in prodrugs of compounds of Formula I The need for such protection may vary depending on the type of remote functional group and the conditions used in the preparation. The need for such protection can be readily determined by one skilled in the art. The use of deprotection / cleavage methods is within the skill of the art. A general description of protecting groups and their use can be found in T, W. Green, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, USA, 1991.

Certain compounds of the present invention contain asymmetric carbon atoms and therefore occur in the form of anantiomers or diastereomers. Diastereometric mixtures

124 · · i i i i i i i i i 9 i 9 9 9 9 9 9 9 9 9 9 9 The individual diastereomers can be separated into their individual diastereomers on the basis of their physico-chemical differences by known methods, for example chromatography and / or fractional crystallization. Enantiomers may be separated by converting the enantiomeric mixture into a diastereometric mixture by reaction with a suitable optically active compound (e.g., an alcohol), then separating the diastereomers and converting the individual diastereomers (e.g., by hydrolysis) to the corresponding pure enantiomers. The present invention includes all such isomers, including diastereomers and enantiomers, as well as mixtures thereof. Some of the compounds of the invention are atropisomers (e.g., substituted biaryls), which are also within the scope of the invention.

Many of the compounds of the invention are acidic and can form salts with pharmaceutically acceptable cations. Some of the compounds of the invention are basic and may form salts with a pharmaceutically acceptable anion. All such salts are within the scope of this invention and can be prepared by conventional methods. For example, they can be prepared simply by mixing the acidic and basic components, usually in a stoichiometric ratio, in an aqueous, non-aqueous or partially aqueous medium, as appropriate. The salts are isolated either by filtration or by non-solvent precipitation followed by filtration, or by evaporation of the solvent or, in the case of aqueous solutions, also by lyophilization.

When the compounds of the invention form hydrates or solvates, they are also within the scope of this invention.

The usefulness of the combinations according to the invention as medicaments suitable for the treatment of said diseases in mammals (e.g. in humans) can be demonstrated by the efficacy of the compounds according to the invention.

125 of the invention in conventional tests and in tests of efficacy and in vivo efficacy tests. in vitro and in vivo described below. These assays also provide a means of comparing the efficacy of the compounds of the invention with those of other known compounds. The results of these comparative tests are useful dosages in mammals, including humans, diseases.

Assay inhibitory activity to determine the level in the treatment of such aldose reductase

The efficacy of an aldose reductase inhibitor can be determined as the amount of aldose reductase inhibitor required to reduce tissue sorbitol, and thus to reduce tissue fructose, using the following procedure.

In male Sprague-Dawley rats, i.v. injection of streptozocin at a dose of 55 mg / kg in citrate buffer pH 4.5 causes diabetes. Rats are fed ad libitum under controlled breeding, temperature and light conditions. After 5 weeks of diabetes, rats are anesthetized with an overdose of pentobarbital, the tissues are harvested rapidly and analyzed for sorbitol and fructose.

Sorbitol levels are analyzed as described in Donald M. Eades et al., Rapid Analysis of Sorbitol, Galactitol, Mannitol, and Myoinositol Mixtures From Biological Sources, Journal of Chromatography, 490, 1-8, 1989.

Fructose in rat tissues is measured enzymatically using a modified procedure described in Ameyama, Methods in Enzymology, 89: 20-29, 1982, whereby ferricyanide

126 · 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

ΦΦ • • · • · • ·

Nahrad ··· replace resazurin, a dye that is reduced to highly fluorescent resorufin. The amount of resorphine fluorescence is in stoichiometry relative to the amount of fructose oxidized by fructose dehydrogenase. The test mixture contained 0.1 ml of neutralized nerve extract, obtained using 6% perchloric acid, in a final volume of 1.5 ml. After incubation for 60 minutes at room temperature in a confined space, the fluorescence of the sample at 560 nm excitation, 580 nm emission with 5 mm slots in the Perkin-Elmer model 650-40 fluorescence spectrometer is determined. The fructose concentration is calculated by comparison with a series of known fructose standards.

Assay for glycogen phosphorylase inhibitory activity

Three different purified glycogen phosphorylase (GP) isoenzymes in which glycogen phosphorylase is in activated and activated state can be prepared by the following procedures. In the further description, these enzymes are referred to as glycogen phosphorylase (or abbreviated GPa), namely glycogen phosphorylase and human liver (HLGPa), glycogen phosphorylase and human muscle (HMGPa), and glycogen phosphorylase and human brain (HBGPa).

Expression and fermentation

HLGP and HMGP cDNA are expressed from plasmid pKK233-2 (Pharmacia Biotech. Inc., Piscataway, New Jersey, USA) in E. coli strain XL-1 Blue (Stratagene Cloning Systems, LaJolla, California, USA). This strain is induced into LB medium (consisting of 10 g tryptone, 5 g yeast extract, 5 g sodium chloride and 1 ml IM sodium hydroxide, per liter of medium) with the addition of 100 mg / liter ampicillin, 100 mg / liter pyridoxine and 600 mg / liter manganese chloride. Cells are grown at 37 ° C to an OD ₅₅₀ = 1.0. The cells are then induced with 1 mM isopropyl-1-thio-β-D

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• ·

-galactoside (IPTG). Three hours after induction, cells are centrifuged and the cell pellet is frozen at -70 ° C. In this state it is stored until subsequent purification.

HBGP and cDNA can be expressed in several ways, for example, as described by Crerar et al., J. Biol. Chem. 270: 13748-13756. The procedure for HBGP expression described in U.S. Pat

Crerar et al., J. Biol. Chem. 270: 13748-13756 is as follows: HBGP cDNA can be expressed from plasmid pTACTAC in E. coli strain 25A6. This strain is inoculated with LB medium (consisting of 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride and 1 ml of 1M sodium hydroxide, relative to 1 liter of medium) with the addition of 50 mg / liter of ampicillin. The cells are grown overnight and then resuspended in fresh LB medium with 50 mg / liter ampicillin. The cell suspension was seeded with a 40-fold volume of LB / amp medium containing 250 µM isopropyl-1-thio-6-D-galactoside (IPTG), 0.5 mM pyridoxine and mM magnesium chloride. Cells are not grown at 22 ° C and centrifuged for 48 to 50 hours. The cell pellet of this state is stored until it can be cleaned up and frozen at -70 ° C and purified.

HLGP cDNA is expressed from plasmid pBlue Bac III (Invitrogen Corp., San Diego, CA) which is co-transfected with BaculoGold linear viral DNA (Pharmingen, San Diego, CA) into Sf9 cells. The recombinant virus is then purified through plaques. Sf9 cells grown in serum-free medium are infected for protein production at a multiplicity of infection (moi) of 0.5 and a cell density of 2 x 10 ⁶ cells / ml. After 72 hours growth at 27 ° C, cells are centrifuged, cell pellets are frozen at -70 ° C and stored at this temperature until purification.

Purification of glycogen phosphorylase expressed in E.coli

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E. coli cells from the pellets described above are resuspended in 25 mM β-glycerophosphate (pH 7.0) with the addition of DTT (0.2 mM),

magnesium chloride (1 mM) and the following proteases: inhibitors pepstatin A 0.7 µg / ml leupeptin 0.5 Aig / inl Phenylmethylsulfonylfluoride (PMSF) 0.2 mM EDTA 0.5 mM Cell lysis is performed pre-processing 200 µg / ml lysozyme and 3 µg / ml DNAase with subsequent processing ultrasound on ice (5 x 1.5 minutes each using 250 ml batches)

an ultrasonic Branson Model 450 cell breaker (Branson Sonic Power Co., Danbury, CT). E. Coli cell lysates are clarified by centrifugation at 35,000 x g for 1 hour and then filtered through 0.45 µm filters. GP in the soluble fraction of the lysate (estimated to be below 1% of total protein) is purified by monitoring the enzymatic activity (as described in the GPa Activity Assay section below, obtained in a series of chromatographic steps described in detail below).

immobilized metal affinity chromatography (IMAC)

This step is based on the method of Luong et al. (Luong et al., Journal of Chromatography (1992) 584, 77-84). 500 ml of the filtered soluble cell lysate fraction (prepared from about 160 g to 250 g of the original cell pellet) is loaded onto a 130 ml IMAC Chelating-Sepharose column (Pharmacia LKB Biotechnology, Piscataway, New Jersey, USA).

129 ······················· pretreated with pH 7 equilibration buffer containing copper chloride (50 mM), γ-glycerophosphate (25 mM), sodium chloride (250 mM) and imidazole (1 mM). Wash the column with equilibration buffer until the ^α 280 value returns to baseline. Then, the sample is eluted from the column with the same buffer, but containing imidazole at 100 mM concentration to remove bound GP and other bound proteins. Fractions containing GP activity were collected (approximately 600 mL) and ethylenediaminetetraacetic acid (EDTA) was added to 0.3 mM, DL-dithiothreitol (DTT) to 0.2 mM, phenylmethylsulfonyl fluoride (PMSF) to 0.2 mM, leupeptin to 0.5 µg / ml and pepstatin A to 0.7 µg / ml. The pooled GP fraction was desalted on a Sephadex G-25 column (Sigma Chemical Company, St. Louis, Missouri, USA) with equilibrated buffer A (buffer A: 25 mM Tris-HCl (pH 7.3), 3 mM DTT) to remove imidazole. . The fraction thus treated is stored on ice until the second chromatography step is carried out.

Chromatography on 5 ^Z -AMP-Sepharose

The desalted pooled GP sample (approximately 600 mL) was then mixed with 70 mL of 5 ^Z -AMP Sepharose (Pharmacia LKB Biotechnology, Piscataway, New Jersey, USA) which was equilibrated with buffer A (see above). The mixture was gently stirred at 22 ° C for 1 hour and then loaded onto the column. Wash the column with Buffer A until the A ₂₈₀ value returns to baseline. GP and other proteins are eluted from the column with buffer B (buffer B: 25 mM Tris-HCl, 0.2 mM DTT and 10 mM adenosine-5 ^from monophosphate (AMP), pH 7.3). The fractions containing GP are pooled after their identification by enzyme activity determination (see below) and the M, approximately 97 kDa GP protein band is visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (

130 ································· · · ··· · ··· · · ··· · ··· ······

The 2D-silver Stain II Daiichi Kit, Daiichi Pure Chemicals Company, Ltd., Tokyo, Japan, and GP were collected. The pooled GP was dialyzed into buffer C (buffer C: 25 mM / J-glycerophosphate, 0.2 mM DTT, 0.3 mM EDTA, 200 mM sodium chloride, pH 7.0) and then stored on ice until the next use.

Prior to use, the GP enzyme is converted from the inactive form expressed in E. coli strain XL-1 Blue (labeled GPb) (Stratagene Cloning Systems, La Jolla, Calif.) To the active form (labeled GPa) by the procedure described in (A) activation. GP below.

Purification of glycogen phosphorylase expressed in Sf9 cells

Sf9 cells from the pellets described above are resuspended in 25 mM M-glycerophosphate (pH 7.0) with the addition of DTT (0.2 mM), magnesium chloride (1 mM) and the following protease inhibitors:

pepstatin A 0.7 Aig / ml leupeptin 0.5 µg / ml phenylmethylsulfonyl fluoride (PMSF) 0.2 mM

EDTA 0,5mM

Cells are lysed by pretreatment using 3 µg / ml DNAase followed by ultrasonic treatment (3x1 min on ice) in batches using a Branson Model 450 ultrasonic cell breaker (Branson Sonic Power Co., Danbury, CT, USA). SF9 cell lysates were clarified by centrifugation at 35,000 x g for 1 hour, and then filtered through 0.45 Zim opener filters. GP in the soluble lysate fraction (estimated at 1.5% total protein) is purified by enzymatic monitoring

131 ······························ Activity (as described in the Testing GPa Activity section below) in samples obtained by a series of chromatographic steps described in more detail below.

Immobilized metal affinity chromatography (IMAC)

The immobilized metal affinity chromatography is carried out as described above. The collected desalted GP is stored on ice until further processing.

To enable GP:

Prior to further chromatography, the inactive enzyme fraction is converted from the inactive form in which it is expressed by Sf9 cells (referred to as GPb) to the active form (referred to as GPa) as described below in section (A) entitled GP Activation.

Anion exchange chromatography

Activation of IMAC purified GPb to GPα is accomplished by reaction with immobilized phosphorylase kinase. The pooled GPa fractions were dialyzed against 25 mM Tris HCl, pH 7.5, containing 0.5 mM DTT, 0.2 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 1.0 µg / ml leupeptin and 1.0 µg / ml. ml of pepstatin A. The sample is placed on an MonoQ anion exchange chromatography column (Pharmacia Biotech. Inc., Piscataway, New Jersey, USA). The column is then washed with equilibration buffer until the A ₂₈₀ returns to baseline. The sample is eluted from the column with a linear gradient of 0 to 0.25M sodium chloride to remove GP and other unbound proteins. The GP containing fractions are eluted at a sodium chloride concentration range of 0.1

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to 0.2 M, as determined by monitoring the eluant for peak protein absorbance at A ₂₈₀ Α · GP protein is then identified by visualizing the M _r approximately 97 kdal GP protein band by polyacrylamide gel by sodium dodecylsulfate (SDS-PAGE) and subsequent silver staining (2D-Silver Stain II Daiichi Kit, Daiichi Pure Chemicals Company, Ltd., Tokyo, Japan) and then collected. The collected GP is dialyzed into buffer (buffer: 25 mM, N, N-bis-2-hydroxyethyl) -2-aminoethanesulfonic acid, 1.0 mM DTT, 0.5 mM EDTA, 5 mM sodium chloride, pH 6.8) and then stored on ice until the next use.

Determination of GP enzymatic activity

A. GP Activation: Convert GPb to GPa

Prior to the determination of the enzymatic activity of GP, the enzyme is converted from an inactive form in which E. coli strain XL-1 (termed GPb) is expressed (Stratagene Cloning Systems, LaJolla, California, USA) to the active form (termed GPa) by phosphorylating GP using phosphorylase kinase as described below. The fraction of the inactive enzyme as expressed in SF9 cells (abbreviated as GPb) is also converted to the active form (abbreviated as GPa) using the following procedure.

Reaction of GP with immobilized phosphorylase kinase

Phosphorylase Kinase (Sigma Chemical Company, St.

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Missouri, USA) was immobilized on Affin Gel 10 Affinity Gel (BioRad Corp. Melvile, New York, USA) according to the manufacturer's instructions. Briefly, the procedure can be described as follows: The phosphorylase kinase enzyme (10 mg) is incubated with washed Affi-Gel beads (1 ml) in 2.5 ml of 100 mM HEPES and 80 mM calcium chloride at pH 7.4 for 4 hours at 4 ° C. Then Affi-Gel beads are washed once with the same buffer, followed by 1 hour blocking with 50 mM HEPES and IM glycine methyl ester pH 8.0 at room temperature. Remove the blocking buffer and replace it with 50 mM HEPES (pH 7.4), 1 mM β-mercaptoethanol and 0.2% sodium azide for storage.

Prior to use for the conversion of GPb to GPa, phosphorylase kinase immobilized on Affi-Geli beads is equilibrated by washing with a buffer used to carry out the reaction with a kinase consisting of β-glycerophosphate (25mM), DTT (0.3mM) and EDTA (0.3mM) and has a pH of 7.8 (kinase assay buffer).

The partially purified inactive GPb obtained by the above 5'-AMP-Sepharose (from E. coli) chromatography or the GPa and GPb mixture obtained by IMAC chromatography (from SF9 cells) is diluted 1: 10 with kinase assay buffer and then mixed with the enzyme phosphorylase kinase described above, which is immobilized on Affi-Geli beads. NaATP (up to 5 mM concentration) and magnesium chloride (up to 6 mM concentration) were added to the mixture. The resulting mixture was gently stirred at 25 ^° C for 30-60 minutes. The sample was separated from the beads and the activation (in%) was determined based on the conversion of GPb to GPa. The degree of activation is estimated by determining GP enzyme activity in the presence and absence of 3.3 mM AMP. Percentage activity of total GP, i. GPa enzyme activity (AMP independent δ activity is then calculated using the following formula:

HLGP - AMP activity

HLGPa share (%) ___________________________

HLGP + AMP activity

134 ν · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Alternatively, the conversion of GPb to GPa can be monitored by isoelectric focusing based on the shift in electrophoretic mobility that is recorded after the conversion of GPb to GPa. Gp samples were analyzed by isoelectric focusing (IEF) using the Pharmacia PfastGel System (Pharmacia Biotech. Inc., Piscataway, New Jersey, USA) using pre-cast gels (pi in the range of 4 to 6.5) and according to the manufacturer's instructions. The bands divided by GPa and GPb are visualized on the gel by subsequent silver staining (2D-silver Stain II Daiichi Kit, Daiichi Pure Chemicals Company, Ltd., Tokyo, Japan). GPa and GPb are identified by comparison with the E. coli GPa and GPb standards, which are obtained in parallel on the same gels as the experimental samples.

B Determination of GPa activity

The curative and prophylactic activity against the aforementioned diseases or conditions can be indirectly determined by the compounds of the invention by determining the effect of the compounds of the invention on the activity of the activated form of glycogen phosphorylase (GPa) using either of the two methods below. Either the glycogen phosphorylase activity is measured in a glucose-1-phosphate formation reaction from the glycogen (cleavage reaction) by monitoring the glucose-1-phosphate content, or measured in a reverse reaction (a synthetic reaction) in which glycogen is synthesized from glucose -l-phosphate, based on the monitoring of inorganic phosphate release. All reactions are performed with three replicates in 96-well microtiter plates and the change in absorbance due to reaction product formation at the wavelength is measured using the MCC / 340 MKII Elisa Reader (Lab Systems, Finland) connected to a Titertech Microplate Stacker ( ICN Biomedical Co. of Huntsville, Alabama, USA).

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To measure the activity of the GPa enzyme in the downstream reaction, glucose-1-phosphate formation from glycogen is monitored by a multienzyme coupled general procedure described by Pesce et al. (Pese MA, Bodourian SH, Harris RC and Nicholson JF (1977) Clinical Chemistry 23 , 1711 to 1717), which is modified as follows: 1 to 100 µg GPa, 10 units phosphoglucomutase and 15 units glucose-6-phosphate dehydrogenase (Boehringer Mannheim Biochemicals, Indianapolis, IN) are diluted to 1 ml with Buffer A (buffer A). pH 7.2, 50 mM HEPES, 100 mM potassium chloride, 2.5 mM ethylene glycol tetraacetic acid (EGTA), 2.5 mM magnesium chloride, 3.5 mM potassium dihydrogen phosphate and 0.5 mM dithiothreitol). 20 µl of the stock is added to 80 µl of Buffer A containing 0.47 mg / ml glycogen, 9.4 mM glucose and 0.63 mM of the oxidized form of nicotinamide adenine diucleotide phosphate (NADP ⁺ ). The test compound is added as a 5 µl solution in 14% dimethylsulfoxide before the enzyme is added. The baseline HLGPα activity value in the absence of inhibitors was determined using the addition of 5-11% 14% dimethylsulfoxide and the total inhibited GPa enzyme activity was determined using the addition of 20 µL of a 50 mM positive control solution, caffeine. The reaction is monitored at room temperature by measuring the conversion of oxidized NADP ⁺ to reduced NADPH at 340 nm.

To measure the activity of the GPa enzyme in a reverse reaction (i.e., a synthetic reaction) in which glucose-1-phosphate produces glycogen and inorganic phosphate, the general method described by Engers et al. (Engers HD, Shechosky, S. and Madsen NB (1970) Can. J. Biochem. 48, 746-754), which is modified as follows: 1 to 100 µg of GPa is diluted to 1 ml with buffer B (buffer B: pH 7) , 2, 50mM HEPES, 100mM potassium chloride, 2.5mM ethylene glycol tetraacetate

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acid (EGTA), 2.5 mM magnesium chloride and 0.5 mM dithiothreitol). 20 µl of stock is added to 80 µl of Buffer B containing 1.25 mg / ml glycogen, 9.4 mM glucose and 0.63 mM glucose-1-phosphate. The test compound is always added as a 5 µl solution in 14% dimethylsulfoxide prior to the addition of the enzyme. Baseline GPa enzyme activity in the absence of added inhibitors was determined by the addition of 5 µl of 14% dimethylsulfoxide, and the total inhibition of GPa enzyme activity was determined by the addition of 20 µL of 50 mM caffeine solution. The mixture is incubated for 1 hour at room temperature and the inorganic phosphate released from glucose-1-phosphate is measured according to the general procedure described by Lanzetta et al. (Lanzetta PA, Alvarez LJ, Reinach PS and Candia OA (1979) Anal. Biochem. 100, 95-97), which is modified as follows: to 100 µl of the enzyme mixture is added 150 µl of 10 mg ammonium molybdenum solution / ml and malachite green (0.38 mg / ml) in 1M hydrochloric acid. The resulting mixture was incubated at room temperature for 20 minutes and the absorbance at 620 nm was measured.

The above tests carried out in the concentration range of the test compounds make it possible to determine the IC ₅₀ value (the concentration of test compound required for 50% inhibition) for the in vitro inhibition of GPa enzyme activity by these test compounds.

Indicative tests for insulin resistance

The combination of the invention can be readily formulated for hypoglycemic agents suitable for clinical use. The hypoglycemic efficacy of the combination of the invention can be determined based on the amount of test glucose lowering compound relative to the vehicle without test compound in males.

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mouse ob / ob. This assay also allows the determination of the approximate minimum effective dose (MED) for the in vivo reduction of glucose levels in mouse plasma using these test compounds.

Because the blood glucose concentration is closely associated with the development of diabetic disorders, the combinations of the invention, by their hypoglycemic effect, prevent, stop and / or regret diabetic disorders.

Five- to eight-week-old male C57 BL / 6J-ob / ob mice (from Jackson Laboratory, Bar Harbor, Maine, USA) are housed in cages for five treatments and treated according to standard methods. After a one week acclimatization period, the animals are weighed and 25 µl of blood is collected from the retro-orbital sinus prior to any treatment. The blood sample is immediately diluted 1: 5 with physiological saline containing 0.025% heparin sodium. The sample obtained is kept on ice until analysis for the metabolite. Animals are assigned to treatment groups such that each group has a similar average plasma glucose concentration. After being assigned to groups of animals for a total of 5 days, a vehicle consisting orally of either 1. a solution of methylcellulose in water at a concentration of 2.5 g / liter without pH adjustment, or 2. a 0.1% solution of Pluronic ( ^R) P105 (block copolymer surfactant, manufactured by BASF Corp., Parsippany, New Jersey, USA) in 0.1% saline without pH adjustment. On day 5, the animals are weighed again and administered the test compound or vehicle alone by oral route. All drugs are administered in a vehicle consisting of either 1. a solution of methylcellulose in water with a 2.5 g / liter concentration without pH adjustment, or 2. a 10% dimethylsulfoxide / 0.1% solution formulation

Pluronic ( ^R ) P105 (BASF Corp., Parsippany, New Jersey, USA) in 0.1% saline, untreated

138 ·· ·· ·· ··· · · · · · ···

9,999 9 · · ··· 9 · · · ··· · ···

9 9 9 9 9 99

99 99 9999 pH. After an additional 3 hours, animals are bled from retroorbital sinus to determine blood levels of the metabolite. The freshly collected samples are centrifuged at 10,000 xg for 2 minutes at room temperature. The supernatant is analyzed for glucose, for example, by the Abbott VP ^ ^R) (Abbott Laboratories, Diagnostics Division, Irving, TX) and VP Super System ^(R Autoanalyzer (Abbott Laboratories, Diagnostics Division, Irving, TX), or Abbott Spectrum CCX ^ ^R ^ Autoanalyzer (Abbott Laboratories, Irving, TX, USA) using the A-Gent ( ^R ) Glucose-UV Test Reagent System (Abbot Laboratories, Irving, TX, USA) (a modification of the method described in Richterrich and Dauwalder, Schweizeriche Mediziniche Wochenschrift, 101, 860 (1971)).

100 mg / 100 ml standards are used in this hexokinase method. computes according to the equation:

Plasma glucose = (mg / 100 ml)

The plasma glucose level is then

The value measured x 8.14 in the sample where the number 8.14 represents the dilution factor adjusted to the plasma hematocrit (assuming the hematocrit is 44%).

In vehicle-administered animals, the hyperglycemic glucose level (e.g., 250 mg / 100 mL or greater) is maintained essentially unchanged when the animals treated with the test compounds at appropriate doses exhibit a substantially reduced glucose level. The hypoglycaemic activity of the test compounds is determined by static analysis (unpaired t-test) using the mean plasma glucose concentration of the test compound and groups,

139 · · ·························· The vehicle, which was administered on day 5 only. The above test, performed with a certain dose range of the test compounds, allows the determination of approximately the minimum effective dose (MED) for in vivo reduction of plasma glucose concentration.

The compounds of the invention can be readily formulated as hyperinsulinemia reversing agents, triglyceride lowering agents, and hypocholestrolemic agents suitable for clinical use. This activity can be determined as the amount of test compound that reduces the concentration of insulin, triglycerides or chloesterol in a male ob / ob mouse relative to a control vehicle without test compound.

Since the blood cholesterol concentration is closely related to the development of cardiovascular, cerebral vascular or peripheral vascular disorders, the combination of the invention, due to their hypocholesterolemic effect, prevents or stops and / or regrets atherosclerosis.

Since blood insulin concentration is closely related to increasing vascular cell growth and increased sodium retention in the kidney (among other effects such as increasing glucose utilization) and since these combinations according to the invention are known to have an effect they prevent the onset of function, induce hypertension, due to their hypoinsulinemic hypertension or to stop or / or regret hypertension.

Since the concentration of triglycerides and free fatty acids in the blood contributes to the total blood lipid level, the combination according to the invention

140 Efficacy in lowering triglycerides and free fatty acids prevents hyperlipidemia or its they stop and / or regret.

Free fatty acids are involved in total blood lipid levels and were independently correlated with insulin sensitivity in various physiological and pathological conditions.

Five- to eight-week-old male C57 BL / 6J-ob / ob mice (from Jackson Laboratory, Bar Harbor, Maine, USA) are housed in cages for five treatments and treated with standard rodent feed ad libitum. After a one-week acclimatization period, the animals are weighed and 25/11 blood is collected from the retro-orbital sinus prior to any treatment. The blood sample is immediately diluted 1: 5 with saline containing 0.025% heparin sodium. The sample obtained is kept on ice until plasma glucose analysis. Animals are assigned to treatment groups such that each group has a similar average plasma glucose concentration. The test compound was administered by oral gavage as a solution of concentration about 0.2 to 20 g / l of either 1) 10% DMSO / 0.1% Pluronic® P105 ^ ^R (Block Copolymer surfactant, BASF Corp., Parsippany, New Jersey, USA) in a 0.1% saline solution without pH adjustment or 2) in a 2.5 g / liter solution of methylcellulose in water, without pH adjustment. Dosing is performed once daily (sid) or twice daily (bid) for a total of 1 to 15 days. Control mice receive only 10% DMSO / 0.1% Pluronic ( ^R ) P105 in 0.1% physiological sodium chloride solution without pH adjustment or methylcellulose solution in water at a concentration of

2.5 g / liter, no pH adjustment.

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Three hours after the last dose administered, the animals are sacrificed by decapitation, and the blood from the trunk is collected in 0.5 ml serum separation tubes containing 3.6 mg of a 1: 1 mixture of sodium fluoride and potassium oxalate in each case. Centrifuge at 10,000 x Og for 10 min at room temperature, and serum supernatant is collected and diluted 1: 1 in volume with a solution of aprotinin (10 IU / ml) in 0.1% sodium chloride solution without pH adjustment.

Diluted serum samples are stored at -80 ° C until analysis. After thawing, diluted serum samples are analyzed for insulin, triglycerides, free fatty acids, and cholesterol. Serum insulin concentration was determined using the Equate ( ^R ) RIA INSULIN kit (two antibody method, according to the manufacturer's instructions) purchased from Binax, South Portland, ME, USA. The inter-assay variance coefficient is less than or equal to 10%. Serum triglyceride levels were determined using the Abbott Vp ( ^R ) and VP Super System ( ^R ) Autoanalyzer (Abbot Laboratories, Irving, TX, USA) or Abbott Spectrum CCX (Abbott Laboratories, Irving, TX, USA). The reagent system A-Gent ( ^R ) Triglycerides Test (Abbot Laboratories, Diagnostics Division, Irving, TX, USA) is used (enzymatic method associated with the use of lipase, modification of the method described by Sampson et al., Clinical Chemistry 21, 1983 (1975)). )). Total serum chloesterol levels were determined using Abbott VP ( ^{R a} and VP Super System ^{R R} Auto Autoanalyzer (Abbot Laboratories, Irving, TX, USA) or Abbott Spectrum CCX ^{R R} ((Abbot Laboratories, Irving, TX, USA). The reagent system A-Gent ( ^R ) Cholesterol Test (enzyme method associated with cholesterol esterase, modification of the method described in Allain et al., Clinical Chemistry 20, 470 (1974)) using 100 and 300 mg standards is used. / 100 ml Concentration

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free fatty acids are determined using a kit from Amano International Enzyme Co., Inc. which is adapted for use with the Abbott Vp ( ^R ) and VP Super System ( ^R ) Autoanalyzer (Abbot Laboratories, Irving, TX, USA) or Abbott Spectrum CCX ( ^R ) (Abbott Laboratories, Irving, TX, USA). Total serum insulin, triglyeride and chloesterol levels are calculated from the following equations

Serum Insulin (AiU / ml) = Sample Value x 2 Serum Triglycerides (mg / 100 ml) = Sample Value x 2 Total Cholesterol (mg / 100 ml) = Sample Value x2 Serum Free Fatty Acid ( = value in the sample x 2 where the number 2 represents the dilution factor.

In animals receiving vehicle only, serum insulin levels (e.g., 275 µl / ml), serum triglycerides (e.g., 235 mg / 100 ml), free serum fatty acids (e.g., 1500 µg / ml) are maintained. and total serum cholesterol (e.g., 190 mg / 100 mL) substantially unchanged when animals treated with the test compounds of the invention generally exhibit reduced serum levels of insulin, triglycerides, free fatty acids, and cholesterol. The total serum lowering activity of test compounds, triglycerides, free fatty acids and cholesterol is determined by statistical analysis (unpaired t-test) using the mean values of insulin, triglycerides, free fatty acids and total cholesterol in the serum group. administration of the test compound and the control group to which only the vehicle was administered.

Efficacy of the compounds of the invention based on

143 9 9 999 9 9999 999

9 9 · 9 9 999

99 99 9 999 protection against ischemia (e.g., cardiac tissue damage) can be demonstrated in vitro using the procedure described by Butwell et al., Am. J. Physiol., 264, H1884 to H1889, 1993, and Allard et al., Am. J Physiol., 1994, 267, H66-74. The experiments were carried out using an isolated rat heart isovolumetric preparation essentially as described above. Normal Sprague-Dawley male rats, Sprague-Dawley male rats who were induced by cardiac aortic lung hypertrophy, acute diabetic male BB / W rats and non-diabetic male BB / W rats (control animals of the same age) were first administered heparin (intraperitoneally). ) and then pentobarbital (intraperitoneally 65 mg / kg. After deep anesthesia, the rapidly cooled solution of retrograde perfusion animals is detected by disappearing the paw reflex, excising the heart and placing sodium chloride. Heart by aorta (within 2 minutes).

The heart rate and ventricular pressure are determined using a latex balloon in the left ventricle using a high pressure tube connected to a pressure gauge. A solution containing sodium chloride (118 mM), potassium chloride (4.7 mM), calcium chloride (1.2 mM), magnesium chloride (1.2 mM), sodium bicarbonate (25 mM) and glucose (11 mM) is used for cardiac perfusion. The perfusion device is well thermostatized to a heart temperature of 37 ° C using thermostatic baths for perfusate and a water duplicator sheath around the perfusion tube. Oxygenation of the perfusate is accomplished using a pediatric hollow fiber oxygenator (Capiax, Terumo Corp., Tokyo,

Japan) right at the heart. The heart is exposed to the perfusion solution with or without the test compound for about 10 minutes or more, followed by 20 minutes of global ischemia and 60 minutes of reperfusion in the absence of the test compound. A comparison is made in the period after ischemia

144 · · 144 144 · 144 Heart rate in control samples with the test compound. During this period, the left ventricular pressure in the control heart is compared with the ventricular pressure in the heart treated with the test compound. At the end of the experiment, perfusion is also performed by staining to determine the proportion of infarct area from the entire area at risk (% IA / AAR), as described below.

The therapeutic effects of the compounds of the invention in preventing cardiac tissue damage that would otherwise occur by ischemic insult can also be demonstrated in vivo as described by Liu et al., Circulation, Vol. 84, no. 1 (June 1991), as specifically disclosed in this specification. In vivo testing is performed to determine the protection of the heart by the test compound relative to a control group receiving only a saline vehicle. To explain, short periods of myocardial ischemia followed by coronary artery reperfusion have been found to protect the heart from subsequent severe myocardial ischemia (Murry et al.,

Circulation 74: 1124-1136,

1986). Cardiac protection, as documented by the reduction in myocardial infarction, can be pharmacologically induced using intravenous adenosine receptor antagonists by intact anesthetized rabbits studied as an in situ model of preconditioned myocardial ischemia (Liu et al., Circulation 84: 350-356, 1991). In vivo testing is performed to determine whether the test compounds are capable of pharmacologically inducing cardiac protection, i. ensure a reduction in the size of myocardial infarction when administered parenterally by intact anesthetized rabbits. The effects of the compounds of this invention can be compared to preconditioning ischemia using an adenosine Al agonist, N ⁶ -1- (phenyl-2R-isopropyl) adenosine (PIA), which is known to pharmacologically induce cardiac protection in intact anesthetized rabbits (study in situ) (Liu et al., Circulation 84: 350-356, 1991). The exact methodology is

145

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described below. Surgical treatment Males of New Zealand rabbit (3 up to 4 kg) anesthetized with sodium salt pentobarbital ( 30 mg / kg i.v.)

By means of a ventral central cervical incision, a tracheotomy is performed and ventilation of the rabbits is effected by a pressure ventilator using 100% oxygen. Drug delivery catheters are placed in the left jugular vein as long as the pressure measuring catheters are placed in the left carotid artery. The heart is exposed to left thoracotomy and a loop (00 silk) is placed around the prominent branch of the left coronary artery. Ischemia is induced by tightly tightening the loop and fastening it with a clamp. The loosening of the loop allows reperfusion of the affected area. Myocardial ischemia is documented by local cyanosis. Reactive hyperaemia is evidence of reperfusion.

protocol

The experiment is started after at least 30 minutes of steady-state arterial pressure and heart rate. Ischemic pretreatment is induced by a double occlusion of the coronary artery, each time for 5 minutes, followed by a 10 minute reperfusion. Pharmacological pretreatment is induced by two infusions of the test compound, for example every 5 minutes, allowing for 10 minutes before further intervention, or by infusion with adenosine agonists, PIA (0.25 mg / kg). After ischemic pretreatment, pharmacological pretreatment is performed or no pretreatment is performed (vehicle pretreatment).

146 ················ 9 9 9 9 9 9999 9 9 9 9

9 9 9 9 9

99 99 9 99 999 control, followed by a 30 minute occlusion of the arthritis followed by a two-hour reperfusion to induce myocardial infarction. The test compound and PIA are dissolved in sodium chloride solution or other suitable vehicle and administered in an amount of 1 to 5 mg / kg.

coloring

Staining is performed as described in Liu et al., Circulation 84: 350-356, 1991. At the end of the two-hour reperfusion period, the heart is quickly removed, plated in a Langendorff apparatus and purged for 1 minute with normal saline solution warmed to body temperature (38 °). C). The suture silk used to form the loop is then tightly retracted to induce arterial reocclusion and a 0.5% suspension of fluorescent particles (1-10 µm) is infused with perfusate (Duke Scientific Corp., Palo Alto, CA, USA) to the whole myocardium stained, except for the area at risk (non-fluorescent ventricular). The heart is rapidly frozen and stored overnight at -20 ° C. The next day, the heart is cut into 2 mm sections, which are stained with 1% triphenyltetrazolium chloride (TTC). Because TTC reacts with color to distinguish the live (stained on dead (unstained infarct) left ventricular section), the infarcted area (uncolored) and the endangered area (without fluorescent particles) are calculated using a calibrated image analyzer to normalize ischemic damage, taking into account differences in area. heart values are expressed as the ratio of infarct area and area at risk (% IA / AAR) All values are expressed as mean values + standard deviation and made with living tissue, possibly red) tissue to tissue. For each use of the preliminary

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statistical comparison using a one-factor ANOVA or an unpaired t-test. Significance is considered at p < 0.05.

The composition and methods of the invention can be tested for usefulness in reducing or preventing ischemic damage to non-cardiac tissues such as the brain or liver, using procedures described in the scientific literature. The compounds of the invention may be administered by a preferred route of administration using a preferred vehicle and at a preferred time of administration, either prior to the ischemic episode, during the ischemic episode, or during all of the following test stages.

The beneficial effect of the compositions and methods of the invention based on the reduction of ischemic brain injury can be demonstrated, for example, in a mammal using the procedure described by Park et al., Ann Neurol. 1988, 24: 543-551.

Briefly, this procedure can be described as follows: adult male Spraque Dawley rats are first anesthetized with 2% halothane and then mechanically ventilated with mixtures of nitrous oxide (70%) and oxygen (30%) containing 0.5 to 1% halothane. A tracheotomy is then made. The working volume of the blower cylinder shall be adjusted so as to maintain the arterial pressure of carbon dioxide at about 4,67 kPa and adequate arterial oxygenation (θ3θ ₂ > · 12 kPa). Body temperature can be monitored using a rectal thermometer and animals can be kept normothermal as necessary by external heat delivery. The animals are then subjected to subtermporal craniectomy to expose the main strain of the left heart cerebral artery (MCA) under an operating microscope. The exposed artery is occluded by microbipolar coagulation, resulting in extensive ischemic lesions in the cerebral cortex and basal ganglia. Mon 3

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At 1 hour of MCA occlusion, rats are deeply anesthetized with 2% halothane and a thoracotomy is performed to infuse heparinized saline into the left ventricular. The effluent is collected by cutting the right atrium. After washing with saline, the tissues are washed with about 200 ml of a 40% solution of formaldehyde, glacial acetic acid and absolute methanol (FAM, 1: 1: 8 by volume), then the animals are decapitated. The head is held in the fixative for 24 hours and then the brain is removed from the tissue, processed, embedded in paraffin wax and cut (about 100 incisions per brain). Sections are digested with heotoxyline-eosin or a combination of cresyl violet and Luxol Fast Blue and examined on a light microscope to identify and quantify ischemic damage using an image analyzer (e.g., Quantimet 720). The volume and area of ischemia is tuned in absolute units (mm ³ and mm ² ) and the percentage of the total area examined is restored. The effect of the compositions and methods of the invention is based on the reduction of MCA occlusion-induced ischemic brain damage by reducing the area or volume of relative or absolute ischemic damage in brain sections of rats in the treatment group compared to brain sections of rats from the placebo-treated control group. .

Alternatively, the methods described in Nakayama et al., Neurology 1988, 38: 1667-1673, Memezawa et al., Stroke 1992, 23: 552-559, Folbergrova, may be used to demonstrate the usefulness of the compositions and methods of the invention in reducing ischemic brain injury. et al., Proc. Natl. Acad. Sci. 1995, 92: 5057-5059 and Gotti et al., Brain Res. 1990, 522: 290-307.

The usefulness of the compositions and methods of the invention in reducing ischemic liver damage in mammals is possible

149 ······························ Demonstrate, for example, using the procedure described in Yokoyama et al., Am. J. Physiol. 1990, 258: G564 to G570.

Briefly, this procedure can be described as follows: adult male Spraque Dawley rats are cheaply anesthetized with pentobarbital sodium (40 mg / kg ip). The animals are then tracheotomized and mechanically ventilated with air from the room. The liver is removed and stored in a controlled environment chamber at a constant temperature of 37 ^° C. Then the liver is washed with a portal vein at a constant pressure of 147 Pa with modified Hemoglobin-free Drawing-Henseleite buffer (118 mM sodium chloride, 4.7 mM potassium chloride, 27 mM). mM sodium bicarbonate, 2.5 mM calcium chloride, 1.2 mM glucose dihydrogen phosphate with the addition of 300 U potassium, heparin).

0.05 mM EDTA and 11 mM The pH of the perfusate is maintained at 7.4 by aerating the buffer with a 95: 5 mixture of oxygen and carbon dioxide. Each liver is washed in a one-way fashion at 20 ml / min for 30 minutes to wash. and equilibration (post-ischemic period), then after a two-hour period of total ischemia and for a further two-hour period of reperfusion under conditions as described for the pre-ischemic period. Aliquots (20 ml) of perfusate are collected during the pre-ischemic period, immediately after the occlusive ischemic period and every 30 minutes of the two-hour reperfusion period. Perfusate samples are assayed for the presence of hepatocellular enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH). These samples are taken to quantitatively reflect the degree of ischemic damage to liver tissue during the procedure. AST, ALT, and LDH activity in the perfusate can be determined by several methods, for example by a reflectometric procedure using a Kodak Ektachem 500 automated analyzer (see Nakano et al., Hepatology 1995, 22: 539-545). Effect of the compositions and methods of the invention in reducing ischemic liver damage

150

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Occlusion-induced induction is recorded by decreasing the release of hepatocellular enzymes immediately after the occlusive period and / or during the postischemic-reperfusion period in the perfused liver of rats from the treatment group compared to the perfused liver of rats from the placebo-treated control group.

Additional procedures and parameters that can alternatively be used to demonstrate the effects of the compositions and methods of the invention in reducing ischemic liver damage can be found, for example, in Nakano et al., Hepatology 1995, 22: 539-545.

The compounds of the invention may be administered by any method by which they can be delivered to the desired tissue (e.g., liver and / or heart tissue). These methods include oral, parenteral, intraduodenal, etc. '. The compounds of the invention are usually administered in a single dose, for example, once daily or in multiple doses.

The combinations of the invention are useful in reducing or minimizing the direct damage to all tissues that may be acceptable for ischemic / reperfusion injury (e.g., heart, brain, lung, kidney, liver, intestine, skeletal muscle and retina) due to an ischemic event (e.g., myocardial infarction). . Thus, the active compounds of the invention are preferably used prophylactically to prevent, i. (perspective or prophylactic) amelioration or arrest, tissue damage (e.g., myocardial tissue) in patients at risk of ischemia (e.g., myocardial ischemia).

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The compounds of the invention are generally administered orally, but parenteral administration (e.g., intravenous, intramuscular, subcutaneous, or intramedullary) is useful, for example, where oral administration is inappropriate for a particular targeting or if the patient is unable to take the drug. . Topical administration may also be indicated, for example, when the patient is suffering from gastrointestinal disorders or if the physician concludes that the most appropriate route of administration of the drug is to the surface of the tissue or organ.

The two different compounds of the invention may be administered simultaneously or sequentially in any order, or may be administered by a single pharmaceutical composition comprising the aldose reductase inhibitor described above and the glycogen phosphorylase inhibitor described above in a pharmaceutically acceptable carrier.

The amount and duration of administration of the compound or compounds of the invention will of course depend on the subject being treated, the severity of the affliction, the mode of administration, and the judgment of the attending physician. Taking into account the wide variability of scientists, the following dosage should be considered as a guide only, and the physician may adjust the dose of the drug to achieve efficacy (e.g., glucose lowering, insulin level) that it deems appropriate for the patient. When considering the desired degree of efficacy to be achieved, the physician must consider a variety of factors, such as the patient's age, past disease, as well as the presence of other diseases (such as cardiovascular disease).

Thus, for example, in one administration mode there is a combination

152 · ♦ · - - - - - - - - - - - - - According to the invention, it can be administered just before heart surgery (for example, within 24 hours) when there is a risk of myocardial ischemia. In an alternative regimen, the compounds may be administered immediately after cardiac surgery (e.g., within 24 hours of surgery) where there is a risk of myocardial ischemia. The compounds of the invention may also be administered on a daily long-term schedule.

Usually, an amount of the combination of the invention is used that is sufficient to produce a corresponding effect based on an increase in insulin sensitivity.

In other words, an amount of a combination of the invention that is sufficient to produce a normal biological effect of insulin at a normal concentration that would be evident in addition to normal pressure and normal glucose tolerance from persistent euglycemia, normoglycemia and normal lipidemia (e.g. triglycerides, cholesterol and free fatty acids).

It is also possible to use the combination of the invention in an amount effective to protect against ischemia.

The aldose reductase inhibitor according to the invention is used in an amount suitable for achieving the effects according to the invention, for example the effect of lowering triglycerides and cholesterol and reversing hyperinsulinemia. The daily effective dose of the aldose reductase inhibitors of the invention is usually in the range of about 0.1 mg / kg to 100 mg / kg, preferably from 0.1 mg / kg to 20 mg / kg, and can be administered in a single dose or several sub-benefits.

An effective daily dose of a glycogen phosphorylase inhibitor according to

153

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of the invention suitable for achieving the desired efficacy, for example in reducing blood glucose, triglycerides, free fatty acids and cholesterol and for reversing the effect on hyperinsulinemia, is in the range of 0.005 to 50 mg / kg, preferably 0.01 to 25 mg / kg and most preferably from 0.1 to 15 mg / kg.

The compounds of the invention are usually administered in the form of pharmaceutical compositions comprising at least one compound of the invention and furthermore a pharmaceutically acceptable vehicle or diluent. Thus, the compounds of the invention may be administered individually or together in any suitable oral, parenteral, rectal or transdermal dosage forms.

For oral administration, the pharmaceutical compositions may be used in pills, capsules, excipients such as calcium phosphate with release agents such as tapioca binders, gum. lubricants, talc.

for soft compositions to be used by the aqueous formulation of the invention to mix with coloring agents, agents, as well as other form powders, citrate solutions, etc. sodium, starch is used, preferably starch and certain complex silicates with is polyvinylpyrrolidone, sucrose tabletting purposes are also magnesium stearate, compositions are also hard gelatin like

On, how

Similar a

of this type contain polyethylene glycols. If suspensions and / or with different emulsifiers diluents, such as suspensions, tablets, tablets containing different calcium carbonate and together with different potato or different gelatin and are also further suitable sodium lauryl sulfate and may be used as a capsule fill. Also preferred are lactose and high meal for oral administration of elixirs, the compounds may be sweetening, flavoring, and / or suspending water, ethanol,

154

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propylene glycol, glycerol and various combinations thereof.

For parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol, as well as sterile aqueous solutions corresponding to water-soluble salts, may be used. Such aqueous solutions may conveniently be buffered and the liquid diluent is isotonized beforehand with sufficient sodium chloride or glucose solution. Such aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injections. Sterile media suitable for these applications can be readily prepared by standard techniques well known to those skilled in the art.

For the purpose of transdermal (e.g. topical) administration, dilute sterile aqueous or partially aqueous solutions (usually about 0.1-5% by weight) can be used. Otherwise, these solutions are similar to the above parenteral solutions.

Methods for making various pharmaceutical compositions with a specified amount of active ingredient are known or obvious to those skilled in the art based on this disclosure. Standard pharmaceutical guides such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easter, PA, USA, 15th edition, 1975) may be used as a suitable guide.

The pharmaceutical compositions of the invention may contain 0.1 to 95%, preferably 1 to 70%, of the compound or compounds of the invention. In any case, it is desirable to ensure that the composition administered comprises a compound or compounds of the invention

155 ·· ΛΛ ··. ··· ··· ························································ In an amount effective to treat the disease or condition of the patient.

Since the invention includes an aspect relating, for example, to the treatment of an insulin resistance condition using a combination of active ingredients that can be administered separately, the invention also provides a combination of separate pharmaceutical form kits. Kit composition: for aldose reductase and for glycogen phosphorylase. to separate compositions, Kit Ingredients.

includes base-base kit separately described described container bottle two above above contains as divided usually contains instructions Form of kit is particularly separate components preferably administered in various forms (e.g. oral and parenteral), compositions in a pharmaceutical inhibitor inhibitor composition or divided foil package, for administration Separately preferred, when dosing at different dosing intervals or when the prescribing physician requires measuring the individual components of the combination.

An example of such a kit is so-called. blister pack. Blister packs are well known in the packaging industry and are widely used to package pharmaceutical unit dosage forms (tablets, capsules, etc.). Blisters usually consist of a sheet of relatively rigid foil-coated material, preferably a transparent plastic material. During the packaging process, the plastic foil is shaped into boxes. The boxes have the size and shape of packaged tablets or capsules. The capsules are then placed in the patches and a sheet of relatively rigid material is attached to the opposite side (with respect to the direction in which the patches were formed) of the plastic film. As a result, the tablets or capsules are enclosed in boxes between the plastic foil

156

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and letter. Preferably, the strength of the sheet is such that the tablets or capsules can be removed from the blister by hand pressing on the box, the opening in the sheet being formed at the location of the box. Tablets or capsules may be selected as described above.

A device to facilitate the removal of the dosage forms from the package should be placed close to the tablets or capsules. This device may take the form of numbers which correspond to the days of the regime in which the tablets are to be taken. As another example, a calendar such as First Week, Monday, Tuesday ... etc., Second Week Monday, Tuesday ... etc. Other variants of such devices are quite clear. The daily dose may be administered in the form of a single tablet or capsule or in the form of several pills or tablets to be taken on a given day. The daily dose of the first compound may consist of a single tablet or capsule, while the daily dose of the second compound may consist of several tablets or capsules and vice versa. This case should be considered in devices facilitating the control of the withdrawal of dosage forms from the package.

According to another particular embodiment, a medicament dispenser (dispenser) adapted to dispense the daily doses at a certain time in a deliberate order is used. Preferably, the dispenser is provided with a device to facilitate withdrawal of dosage forms if this helps to increase compliance with the regimen. An example of a device for facilitating the control of the withdrawal of dosage forms is a mechanical counter of separate daily doses. Another example of such a device is a battery-powered microchip memory coupled to a liquid crystal display or an audio memory signal that shows, for example, the date the last dose was taken and / or reminds when the next dose is to be taken.

157

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The compounds of the invention, alone or in combination with other compounds, will be administered in the form of conventional formulations. The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way. The term active ingredient refers to a compound or compounds of the invention and may refer to an aldose reductase inhibitor or a glycogen phosphorylase inhibitor or a combination of the two.

Formulation 1: Gelatin capsules

Hard gelatine capsules are prepared using the following ingredients:

Ingredient Quantity (mg / capsule)

active ingredient 0.25 until 100 Starch, NF 0 until 650 starch powder 0 until 50 liquid silicone with a kinetic viscosity of 350.10 ^- ^ m ^ .s -1 0 until 15

Using the following ingredients, a tablet formulation is prepared:

Formulation 2: Tablets

component

Amount (mg / tablet) active ingredient

0.25 to 100

158

Microcrystalline cellulose sublimated silica acid microcrystalline cellulose stearic

200

until 650 until 650 until 15

The ingredients are mixed and the resulting mixture is compressed into tablets.

Tablets, each containing 0.25 to 100 mg of the active ingredients, may alternatively be prepared as follows:

Formulation 3: Tablets

component

Amount (mg / tablet) active ingredient starch microcrystalline cellulose polyvinylpyrrolidone (as a 10% solution in water) sodium carboxymethylcellulose magnesium stearate talc

0.25 to 100

4.5

0.5

The active ingredients, starch and cellulose are passed through a sieve with a mesh size of 355 µm and mixed thoroughly. The resulting powder was mixed with a polyvinylpyrrolidone solution and passed through a 1400 .mu.m sieve. The granules thus prepared are dried at 50 to 60 ° C and passed through a sieve

159 ·· ··

·. # _ JU ... - ··· ··· ······················································· A mesh size of 1000 µm. Sodium carboxymethylcellulose, magnesium stearate and talc are added to the granules, sieved through a 250 µm sieve. The ingredients are blended and tablets are compressed on a tabletting machine.

A suspension containing 0.25 to 100 mg of active ingredient per 5 ml dose is prepared as follows:

Formulation 4: suspension

component

Quantity (mg / 5 ml)

active ingredient 0.25 to 100 mg sodium carboxymethylcellulose 50 mg syrup 1.25 mg benzoic acid solution 0,10 ml flavor or flavor enhancer q. in. coloring substance q. in. purified water to 5 ml

The active ingredient is passed through a sieve having a mesh size of 355 Aim and mixed with sodium carboxymethylcellulose and syrup to form a smooth paste. The benzoic acid solution, flavor or flavor, and coloring agent are diluted with water and added to the paste with stirring. Water is added to the resulting mixture in sufficient quantity to achieve the desired volume.

The aerosol solution is prepared using the following components:

160

Formulation 5: aerosol

· · · · · · • • • • • • • • · • • · · · • • • · • • • • • · · • • ···· · • • • · · · · · · • ·· ·

component

Quantity (% w / w)

active ingredient 0.25 ethanol 25.75 propellant 22 (chlorofluoromethane) 70.00

The active ingredient is mixed with ethanol and the resulting mixture is added to a portion of the propellant 22, the resulting mixture is cooled to 30 ° C and transferred to a filling machine. The stainless steel container is filled with the required amount and the contents are diluted with the remaining propellant. The vessel is then equipped with a valve.

The laces are prepared as follows:

Formulation 6: Lace f Component Quantity (mg / suppository) active ingredient 250 glycerides of saturated fatty acids 2000

The active ingredient is passed through a 250 µm sieve and suspended in saturated fatty acid glycerides melted using minimal heat. The mixture is then poured into a lace mold with a minimum capacity of 2 g and allowed to cool.

161

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The intravenous formulation is prepared as follows:

Formulation 7: Intravenous solution

Component Amount active ingredient isotonic saline solution

The solution of the above ingredients was injected intravenously at a rate of about 1 ml / min.

mg

1000 ml is given to the patient

Combinations of agents are also included within the scope of the active ingredient.

Claims (53)

PATENT CLAIMS II · e ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ·· ·· · ·· CLAIMS 1. A pharmaceutical composition comprising a therapeutically effective amount a. aldose reductase inhibitor, b. glycogen phosphorylase inhibitor and c. a pharmaceutically acceptable carrier. 2. A pharmaceutical composition according to claim 1 wherein the aldose reductase inhibitor is 3,4-dihydro-4-oxo-3- [5- (trifluoromethyl) -2-benzothiazolyl] methyl] -1-phthalazineacetic acid or a pharmaceutically acceptable salt thereof. sol. The pharmaceutical composition according to claim 2, wherein the glycogen phosphorylase inhibitor is β (1S) -benzyl- (2R) -hydroxy-3 - ((3S) -hydroxypyrrolidin-1-yl) -3-oxopropyl] amide 5- chloro-1H-indole-2-carboxylic acid, 5-chloro-1H-benzyl-3 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide; indole-2-carboxylic acid, RLS) - ((R) -hydroxydimetylkarbamoylmetyl) -2-phenylethyl amide 5-chloro-1H-indole-2-carboxylic acid, 5-Chloro-1H-indole-2-carboxylic acid C (1S) - ((R) -hydroxymethoxymethylcarbamoyl) methyl) -2-phenylethyl 1J, [(1S) - ((R) -hydroxy-C (2) 5-chloro-1H-indole-2-carboxylic acid-hydroxyethyl) methylcarbamoyl (methyl) -2-phenylethyl ester, 163 5-Chloro-1H-indole-2-carboxylic acid [(1S) -benzyl-2- (3-hydroxyiminopyrrolidin-1-yl) -2-oxoethyl] amide, 5-Chloro-1H-indole-2-carboxylic acid 2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2-oxoethyl-3-amide, E 2 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) 5-Chloro-1H-indole-2-carboxylic acid -2-oxoethyl amide, N - (1S) -benzyl-3 - ((cis) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide 5- 5-Chloro-1H-indole-2-carboxylic acid chloro-1H-indole-2-carboxylic acid [(1S) -benzyl-2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2-oxoethyl] -amide 5-Chloro-1H-indole-2-carboxylic acid 2- (1,1-dioxo-thiazolidin-3-yl) -2-oxo-ethyl-amide, 5-Chloro-1H-indole-2-carboxylic acid (1S) - (4-fluoro-benzyl) -2- (4-hydroxypiperidin-1-yl) -2-oxoethyl-7-amide, E (1S) -benzyl-2- ( 5-Chloro-1H-indole-2-carboxylic acid (3RS) -hydroxypiperidin-1-yl) -2-oxoethyl amide, 5-Chloro-1H-indole-2-carboxylic acid 2-oxo-2- ((1RS) -oxothiazolidin-3-yl) ethyl) amide, or 5-Chloro-1H-indole-2-carboxylic acid (1S) -benzyl-2- (3-hydroxy-azetidin-1-yl) -2-oxo-ethyl] -amide. 4. The pharmaceutical composition of claim 3 wherein the amount of aldose reductase inhibitor is about 0.1 mg / kg to about 20 mg / kg. 164 • e. - - - - - - - - - - - - - - - - - The pharmaceutical composition of claim 4, wherein the amount of glycogen phosphorylase inhibitor is about 0.1 mg / kg to about 15 mg / kg. 6. A method for treating a mammal afflicted with an insulin resistance condition, comprising administering to said mammal a therapeutically effective amount. a. aldose reductase inhibitor a b. a glycogen phosphorylase inhibitor. The method of claim 6, wherein the insulin resistance state is diabetes, hyperinsulinemia, impaired glucose tolerance, hyperglycemia and / or post-meal hyperlipidemia, type II diabetes, altered body composition, reduction of substantially non-muscle tissue mass fat, obesity, hypertension, dyslipidemia, atherosclerosis, tissue ischemia, cardiovascular disease, obesity, syndrome X, pregnancy, infectious condition, uremia, hyperadrogenism, hypercortisolemia or other condition of excess adrenocortical hormones, or acromegaly growth, polymorphism. 8th The method of claim 6, characterized in that the insulin resistance state is diabetes. The method of claim 8, wherein the aldose reductase inhibitor is 3,4-dihydro-4-οχο-3-f [5- (trifluoromethyl) -2-benzothiazolyl] methyl 1 H -1-phthalazineacetic acid or a pharmaceutically acceptable salt thereof. . 165 Jl · ··· ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · The method of claim 9, wherein the glycogen phosphorylase inhibitor is β (1S) -benzyl- (2R) -hydroxy-3 - ((3S) -hydroxypyrrolidin-1-yl) -3-oxopropyljamide 5-chloro- 5-Chloro-1H-indole-2-carboxylic acid N - (1S) -benzyl-3 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide 2-carboxylic acid, 5-chloro-1H-indole-2-carboxylic acid [(1S) - ((R) -hydroxydimethylcarbamoylmethyl) -2-phenylethyl] -amide, (S) - ((R) -hydroxymethoxymethylcarbamoyl) methyl) 5-Chloro-1H-indole-2-carboxylic acid, 2-phenylethyl amide, [(1S) - ((R) -hydroxy- [(2-hydroxyethyl) methylcarbamoyl] methyl) -2-phenylethyl] amide 5-chloro-1H- 5-Chloro-1H-indole-2-carboxylic acid, indole-2-carboxylic acid, N - (1S) -benzyl-2- (3-hydroxy-imino-pyrrolidin-1-yl) -2-oxo-ethyl-amide; 5-Chloro-1H-indole-2-carboxylic acid 4-dihydroxypyrrolidin-1-yl) -2-oxoethyl amide 5-Chloro-2-oxo-pyrrolidin-1-yl) -2-oxoethyl amide -1H-indole-2-carboxylic acid, E (1S) -benzyl-2- (cis-3,4-dihydrate) 5-Chloro-1H-indole-2-carboxylic acid oxypyrrolidin-1-yl) -2-oxoethyl] amide 5-Chloro-1H-indole 2- (1,1-dioxothiazolidin-3-yl) -2-oxoethyl amide 5-Chloro-1H-indole-2-carboxylic acid (2S) -benzyl-3 - ((cis) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl] -2-carboxylic acid, 166 • · .... • • · • ·· • · • • · • · · · · • • • · • * • · • · · • • ···· • · · e • · ·· • · • · • · ·· • • • · ·· * ·· · 5-Chloro-1H-indole-2-carboxylic acid (1S) - (4-fluoro-benzyl) -2- (4-hydroxypiperidin-1-yl) -2-oxo-ethyl] -amide, £ (1S) -benzyl-2 5-Chloro-1H-indole-2-carboxylic acid - ((3RS) -hydroxypiperidin-1-yl) -2-oxoethyl] -2-oxo-2 - ((1RS) -oxothiazolidin-3-yl) ethyl] - 5-chloro-1H-indole-2-carboxylic acid amide, or 5-chloro-1H-indole-2-carboxylic acid [(1S) -benzyl-2- (3-hydroxyazetidin-1-yl) -2-oxoethyl] amide. 11. The method of claim 10, wherein the amount of aldose reductase inhibitor is about 0.1 mg / kg to about 20 mg / kg and the amount of glycogen phosphorylase inhibitor is about 0.1 mg / kg to about 10 mg. / kg. 12. The method of claim 11, wherein the mammal is a male or female human. The method of claim 6, wherein the insulin resistance state is obesity. 14. The method of claim 6, wherein the insulin resistance state is polycystic ovaries. The method of claim 6, wherein the insulin resistance state is Syndrome X. 167 · · ··. . .M ... >. .. • • • • • • • • · · · • • • · • • • · • · • ···· · • • • · • • • · · · · · · • 16. The method of claim 1, wherein the resistance state 6, wherein insulin is hypertension. 17. A kit comprising a. a therapeutically effective amount of an aldose reductase inhibitor and a pharmaceutically acceptable carrier in a first unit dosage form, b. a therapeutically effective amount of a glycogen phosphorylase inhibitor and a pharmaceutically acceptable carrier in a second unit dosage form, and c. a container for said first and second dosage forms. Kit according to claim 17, characterized in that the aldose reductase inhibitor is 3,4-Dihydro-4-oxo-3- [5- (trifluoromethyl) -2-benzothiazolyl] methyl] -1-phthalazineacetic acid or a pharmaceutically acceptable salt thereof. Kit according to claim 18, characterized in that the glycogen phosphorylase inhibitor is β (1S) -benzyl- (2R) -hydroxy-3 - ((3S) -hydroxypyrrolidin-1-yl) -3-oxopropyl-5-chloro- 5-Chloro-1H-indole-1H-indole-2-carboxylic acid f (1S) -benzyl-3 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide 2-carboxylic acid, 5-Chloro-1H-indole-2-carboxylic acid C (1S) - ((R) -hydroxydimethylcarbamoylmethyl) -2-phenylethyljamide, 168 • · · • · · • • · • - · · • ·· • • • • · · · • · • · • • • • • • · 9 · · ···· • · • • • • • · • · • • • • · · · · · · • · · · · · 5-Chloro-1H-indole-2-carboxylic acid ((S) - ((R) -hydroxymethoxymethylcarbamoyl) methyl) -2-phenylethyl iamide; 5-Chloro-1H-indole-2-carboxylic acid -2-phenylethyl-amide, 5-chloro-1H-indole-2 (3-hydroxyiminopyrrolidin-1-yl) -2-oxoethyl-amide 2-carboxylic acid, 5-chloro-1H-indole-2-carboxylic acid, 2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2-oxoethyl] -amide, £ 2 - ((3S, 4S) 5-chloro-1H-indole-2-carboxylic acid-dihydroxypyrrolidin-1-yl) -2-oxoethyl) amide, [(1S) -benzyl-2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2 5-Chloro-1H-indole-2-carboxylic acid-oxoethyl-amide, η 2- (1,1-dioxothiazolidin-3-yl) -2-oxoethyl, 5-chloro-1H-indole-2-carboxylic acid amide; 5-Chloro-1H-indole-2-carboxylic acid 1S) -benzyl-3 - ((cis) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide, S (1S) - (4- 5-Chloro-1H-indole-2-carboxylic acid fluorobenzyl) -2- (4-hydroxypiperidin-1-yl) -2-oxoethyl amide, E (1S) -benzyl-2 - ((3RS) -hydroxy) 5-Chloro-1H-indole-2-carboxylic acid piperidin-1-yl) -2-oxoethyl amide 5-Chloro-1H-indole 2-oxo-2 - ((1RS) -oxothiazolidin-3-yl) ethyl amide -2-carboxylic acid, or E (1S) -benzyl-2- (3-hydroxyazetidin-1-yl) -2-oxoethyl amide 5-chloro-1H-indole-2-carboxylic acid. 169 • · • · • · • e · · • ··· • · • · • · • · · • • · ···· φ • · • · • · • · · • · • • · • • • • · ·· • · • · • · ·· • • · • · · The kit of claim 19, wherein the amount of the aldose reductase inhibitor is about 0.1 mg / kg to about 20 mg / kg. The kit of claim 20, wherein the amount of glycogen phosphorylase inhibitor is about 0.1 mg / kg to about 15 mg / kg. 22. A pharmaceutical composition for increasing sensitivity to insulin contains in mammals, characterized by because of a. quantity first compound which is a inhibitor aldose reductase, and b. quantity second compound which is a inhibitor glycogen phosphorylase, wherein the amount of the first compound alone and the amount of the second compound alone is insufficient to increase insulin sensitivity when administered alone and the combined effect of the amount of the first compound and the amount of the second compound is greater than the sum of insulin sensitizing effects that can be achieved with the amount of the first and second the compounds in particular, and a pharmaceutically acceptable diluent or carrier. The pharmaceutical composition of claim 22, wherein the aldose reductase inhibitor is 3,4-dihydro-4-oxo-3-LL 5 - (trifluoromethyl) -2-benzothiazolyl] methyl] -1-phthalazineacetic acid or a pharmaceutically acceptable salt thereof. 170 · B e e e B e · · e e e 24. The pharmaceutical composition of claim 23, wherein the glycogen phosphorylase inhibitor is β (1S) -benzyl- (2R) -hydroxy-3 - ((3S) -hydroxypyrrolidin-1-yl) -3-oxopropylamide 5- chloro-1H-indole-2-carboxylic acid, 5-chloro-1H-indole, [(1S) -benzyl-3 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl] -amide 5-Chloro-1H-indole-2-carboxylic acid, 2-phenylethyl-2-carboxylic acid, N (1S) - ((R) -hydroxydimethylcarbamoylmethyl) -2- (1S) - ((R) -hydroxymethoxymethylcarbamoyl) methyl 5-Chloro-1H-indole-2-phenylethyl amide 5-chloro-1H-indole-2-carboxylic acid, (1S) - ((R) -hydroxy-β (2-hydroxyethyl) methylcarbamoyl) methyl) -2-phenylethyl amide 5-Chloro-1H-indole-2-carboxylic acid, N - (1S) -benzyl-2- (3-hydroxyiminopyrrolidin-1-yl) -2-oxoethyl] -amide, N-2- (cis-3,4) 5-Chloro-1H-indole-2-carboxylic acid-dihydroxypyrrolidin-1-yl) -2-oxoethyl amide 5-Chloro-2 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) -2-oxoethyl amide 1H-indole-2-carboxylic acid 5-Chloro-1H-indole-2-carboxylic acid (1S) -benzyl-2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2-oxoethyl amide, 5-Chloro-1H-indole-2-carboxylic acid (1S) -benzyl-3 - ((cis) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide, 171 9 9 9 9 99 99 9 9 9 9 9 9 9999999 2- (1,1-dioxothiazolidin-3-yl) -2-oxoethyl 3-amide 5-chloro 5-Chloro-1H-indole-2-carboxylic acid, 1 (S) - (4-fluoro-benzyl) -2- (4-hydroxypiperidin-1-yl) -2-oxo-ethyl-amide, -1H-indole-2-carboxylic acid, 5-Chloro-1H-indole-2-carboxylic acid, 5-chloro-1H-indole-2-carboxylic acid, (2S) -benzyl-2 - ((3RS) -hydroxypiperidin-1-yl) -2-oxo-2 - ((1RS) - 5-chloro-1H-indole-2-carboxylic acid oxothiazolidin-3-yl) ethyl amide or 5-chloro-f (1S) -benzyl-2- (3-hydroxyazetidin-1-yl) -2-oxoethyl amide -1H-indole-2-carboxylic acid. 25. The pharmaceutical composition of claim 24, wherein the amount of the aldose reductase inhibitor is about 0.1 mg / kg to about 20 mg / kg. 26. The pharmaceutical composition of claim 25, wherein the amount of glycogen phosphorylase inhibitor is about 0.1 mg / kg to about 15 mg / kg. 27. A method of increasing insulin sensitivity in a mammal suffering from an insulin resistance state, comprising administering to said mammal. a. an amount of the first compound which is an aldose reductase inhibitor; and b. the amount of the second compound, which is a glycogen phosphorylase inhibitor, 172 wherein the amount of the first compound alone and the amount of the second compound alone is insufficient to increase insulin sensitivity when administered alone, and the combined effect of the amount of the first compound and the amount of the second compound is greater than the sum of insulin sensitizing effects that can be achieved with the amount of the first and the second compound separately. The method of claim 27, wherein the aldose reductase inhibitor is 3,4-dihydro-4-oxo-3- 5- (trifluoromethyl) -2-benzothiazolyl methyl-1-phthalazineacetic acid or a pharmaceutically acceptable salt thereof. 29. The method of claim 28 wherein the glycogen phosphorylase inhibitor is 5-Chloro-1H-indole-2-carboxylic acid (1S) -benzyl- (2R) -hydroxy-3 - ((3S) -hydroxypyrrolidin-1-yl) -3-oxopropyl-amide, 5-Chloro-1H-indole-2-carboxylic acid (1S) -benzyl-3 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3-oxopropyl-amide, 5-Chloro-1H-indole-2-carboxylic acid (1S) - ((R) -hydroxydimethylcarbamoylmethyl) -2-phenylethyl iamide, 5-Chloro-1H-indole-2-carboxylic acid (1S) - ((R) -hydroxymethoxymethyl-carbamoyl) -methyl-2-phenylethyl-amide, 5-Chloro-1H-indole-2-carboxylic acid (1S) - ((R) -hydroxy- [(2-hydroxyethyl) methylcarbamoyl] methyl) -2-phenylethyl] amide, f (1S) -benzyl-2- 5-Chloro-1H-indole-2-carboxylic acid (3-hydroxyiminopyrrolidin-1-yl) -2-oxoethyl amide, 173 -vr · · · vr vr _ _ _ _ _ _ _ vr vr vr vr vr vr vr vr vr vr 5-Chloro-1H-indole-2-carboxylic acid 2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2-oxoethyl] amide, [2 - ((3S, 4S) 5-Chloro-1H-indole-2-carboxylic acid-2-oxo-ethyl-pyrrolidin-1-yl) - (1S) -benzyl-2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2- 5-Chloro-1H-indole-2-carboxylic acid oxoethyl amide, E 2- (1,1-dioxothiazolidin-3-yl) -2-oxoethyl 5-chloro-1H-indole-2-carboxylic acid amide, E (1S) 5-Chloro-1H-indole-2-carboxylic acid 5-chloro-1H-indole-2-carboxylic acid-2-benzyl-3 - ((cis) -dihydroxypyrrolidin-1-yl) - (2R) - (4S) 5-Chloro-1H-indole-2-carboxylic acid -2- (4-hydroxypiperidin-1-yl) -2-oxoethyl amide, E (1S) -benzyl-2 - ((3RS) -hydroxypiperidin-1-yl) 5-Chloro-1H-indole-2-oxo-2 - ((1RS) -oxothiazolidin-3-yl) ethyl} amide 5-Chloro-1H-indole-2-carboxylic acid -2-oxoethyl amide -carboxylic acid, or [(1S 5-Chloro-1H-indole-2-carboxylic acid 1-benzyl-2- (3-hydroxyazetidin-1-yl) -2-oxoethyl amide. 30. The method of claim 29, wherein the amount of aldose reductase inhibitor is about 0.1 mg / kg to about 20 mg / kg. 31. The method of claim 30, wherein the amount of glycogen phosphorylase inhibitor is about 0.1 mg / kg to about 15 mg / kg. 174 • · 32. The method of claim 31, wherein the mammal is a male or female human. 33. A method for reducing tissue damage induced by ischemia, comprising administering to a mammal in need of such treatment a therapeutically effective amount. a. an aldose reductase inhibitor; and b. a glycogen phosphorylase inhibitor. 34. The method of claim 33, wherein the tissue is heart, brain, liver, adrenal, lung, intestine, skeletal muscle, spleen, pancreas, nerve, spinal cord, retina, vasculature, or intestinal tissue. 35. The method of claim 34 wherein the aldose reductase inhibitor is 3,4-dihydro-4-oxo-3- 5- (trifluoromethyl) -2-benzothiazolyl methyl-1-phthalazineacetic acid or a pharmaceutically acceptable salt thereof. 36. The method of claim 35, wherein the glycogen phosphorylase inhibitor is 5-Chloro-1H-indole-2-carboxylic acid C (1S) -benzyl) - (2R) -hydroxy-3 - ((3S) -hydroxypyrrolidin-1-yl) -3-oxopropyl] amide, r (1S) -benzyl -3 - ((3S, 4S) -dihydroxypyrrolidin-1-yl) - (2R) 175 5-Chloro-1H-indole-2-carboxylic acid-hydroxy-3-oxopropyl-amide, [(1S) - ((R) -hydroxydimethylcarbamoylmethyl) -2-phenylethyl] 5-chloro-1H-indole-2-carboxylic acid amide acid, 5-Chloro-1H-indole-2-carboxylic acid (1S) - ((R) -hydroxymethoxymethylcarbamoyl) methyl) -2-phenylethyl amide, 5-Chloro-1H-indole-2-carboxylic acid (1S) - ((R) -hydroxy-L (2-hydroxy-ethyl) -methyl-carbamoyl-methyl) -2-phenylethyl-amide, 5-Chloro-1H-indole-2-carboxylic acid (1S) -benzyl-2- (3-hydroxy-imino-pyrrolidin-1-yl) -2-oxo-ethyl-amide, η 2- (cis-3,4-dihydroxypyrrolidin-1-yl) 5-Chloro-1H-indole-2- (2-oxo-ethyl) -amide, 5-chloro-1H-indole-2- (2S, 4S) - (3S, 4S) -dihydroxypyrrolidin-1-yl) -2-oxo-ethyl-amide carboxylic acid, 5-Chloro-1H-indole-2-carboxylic acid (1S) -benzyl-2- (cis-3,4-dihydroxypyrrolidin-1-yl) -2-oxoethyl amide, η 2- (1,1-dioxothiazolidine-) 3-yl) 5-chloro-1H-indole-2-carboxylic acid -2-oxoethyl amide, E (1S) -benzyl-3 - ((cis) -dihydroxypyrrolidin-1-yl) - (2R) -hydroxy-3- 5-Chloro-1H-indol-2-yl (5-chloro-1H-indole-2-carboxylic acid oxopropyl) -amide, E (5S) - (4-fluorobenzyl) -2- (4-hydroxypiperidin-1-yl) -2-oxoethyl amide 5-chloro-1H-indole-2-carboxylic acid [(1S) -benzyl-2 - ((3RS) -hydroxypiperidin-1-yl) -2-oxoethyl] -amide, 176 · 2 2 999 999 999 2 999 2 2 2 2 2 2 2 999 999 2 2 2 2 999 2 2 2 2 999 999 2 999 999 5-Chloro-1H-indole-2-carboxylic acid -2 - ((1RS) -oxothiazolidin-3-yl) ethyl amide, or E (IS) -benzyl-2- (3-hydroxyazetidin-1-yl) 5-Chloro-1H-indole-2-carboxylic acid -2-oxoethyl amide. 37. The method of claim 36, wherein the amount of aldose reductase inhibitor is about 0.1 mg / kg to about 20 mg / kg and the amount of glycogen phosphorylase inhibitor is about 0.1 mg / kg to about 15 mg. / kg. 38. The method of claim 38, wherein the mammal is a male or female human. 39. The method of claim 38, wherein the tissue is heart tissue. 40. The method of claim 38, wherein the tissue is brain tissue. 41. The method of claim 38, wherein the tissue is liver tissue. 42. The method of claim 38, wherein the tissue is adavine tissue. The stage method of claim 38, wherein the tissue is lung tissue. 44th 10. The method of claim 38, wherein the tissue is intestinal tissue. 177 45th Method according to claim 38, characterized in that ‘·· • · • · • · ’·· - * • · · · • · · • · • · • · •. • • · ···· • · · • · • · • · · • · • • · • • • • · ·· • · ·· • * ·· • • · · · · in that the tissue is skeletal muscle tissue. 46. The method of claim 38, wherein the UCI satellite ⁴ wherein said tissue is spleen tissue. to 47. The method of claim 38, wherein the tissue is pancreatic tissue. 48. The method of claim 38, wherein the tissue is retinal tissue. 49. The method of claim 38, wherein the combination is administered prophylactically. 50. The method of claim 38, wherein the combination is administered prior to cardiac surgery. 51. The method of claim 33, wherein the tissue injury induced by ischemia * occurred during organ transplantation. * 52. A method of treating a mammal suffering from an insulin resistance condition, comprising administering to said mammal the composition of claim 1. 53. A method of reducing tissue damage caused by ischemia, comprising administering to a mammal in need of such treatment a composition according to claim 1.